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1 TheCharacterizationofOrthologousCG14561, CG7139 PA,CG7139 PB,CG7133,CG7130andthe IneffectualExclusionofDrosophilamelanogaster ExonicSequenceinRpLP0inDrosophilaerecta GenomicsEducationPartnership Fosmid16 HarryQuedenfeld Dataandtextfromthispaperisallowedtobeincludedinpublications Overview Thearearepresentedbymyfosmidstartsatapproximatelybasepairnumber22,058,504andproceeds44,625 basepairsuntilabout22,103,128indrosophilamelanogaster,thoughfosmid16is40,000bpind.ere.,which showsthateitherthelatterhasdeletionsortheformerinsertions.thefigurebelowshowsthegene containing region,astherestofdna3 ofmsopacontainsnogenes.theintentionofthisprojectwastodeterminegenes withinmyfosmidofdrosophilaerectaandprobablefunctionofthosegenesbasedond.mel.asareference genome.thetranscriptsofthegenescg14561,cg7139,cg7133,cg7130,rplp0,sfp79bandmsopawerefound tohavehomologousregionswithmyfosmidbyuseofblast2,andallconfirmedgeneshadthesameamountof exonsastheirorthologousgenesind.mel(rplp0isanexceptionduetogenechecker sinsufficiencytodetect untranslatedexons).sfp79bandmsopaarethoughttobepseudogenesbecauseoftheirshortpeptidelengthand highdegreeofmutationfromd.mel.evidencefromd.mel.supportsthatsimilargeneswithsimilarfunctional proteinsarefoundind.ere.forallgenesexceptsfp79bandmsopa.rplp0 sfirstexonandaportionofthesecond arepartofthe3 untranslatedregionind.mel.rplp0ind.mel.doesnothaveastartcodoninitsfirstexon,but inthemiddleofitssecond.ind.ere.,however,thereisamutationwhichcausesforaprematurestartand readingofaprematurestopcodon.ifthisnormallyuntranslated1 st exonwastranscribedandtranslated,the proteinwouldbenonfunctionalandapseudogene.ifthe1 st exonisnottranscribed,thentherplp0proteinind. ere.is98%identicaltod.mel. s,whichisexpectedforaribosomalprotein.genescg14561,cg7139andcg7130 ind.ere.havenosignificantdifferencesfromtheird.mel.ortholog.cg7133ind.ere.issimilarlengthbutshows a55%identitywithd.mel. s,soitprobablyhasadifferentfunctionind.ere.thisproteinisnothighlyconserved withincloserelativesofd.ere.andisstillthoughttobeagene,whichevidencesitsprobabilitytobeageneind. ere.aswell.inthespeciesdrosophilasimulans,drosophilasechelliaanddrosophilayakuba,cg7133isnothighly conservedbutisstillaputativegene,thusthisisalsoageneind.ere.thegenesfp79bisapseudogenebecauseit

HarryQuedenfeld Genomics Dr.Moss isonly4aminoacidsinlengthduetoamutationthatcodesforanearlystartcodon,whichisfollowedbyastop codon.

2 HarryQuedenfeld Genomics Dr.Moss isonly4aminoacidsinlengthduetoamutationthatcodesforanearlystartcodon,whichisfollowedbyastop codon.msopaisalsothoughttobeapseudogenebecauseitisonly64aminoacidslongandhasalargedeletion whencomparedtod.mel. Dere/GG13189 RB Genes CG14561/NM_ CG14561 PAisthenameoftheorthologinmelanogastertothegenefoundinfosmid16ofD.ere.Ithasasingle isoform,cg14561 PA.Thisgenecodesfrom andthestopcodonispresentat infosmid16.It isasingleexongeneandcodesintheplusdirection.itisageneind.mel.andthusisprotein coding,yetits functionandgeneontologyisunknown.theblastpbetweentheproteinforthegeneiannotatedind.ere.and theknowngeneind.mel.yieldsasignificante valueof3e 100andwith89%identity,showingthattheyare ratherhomologous.thefunctionind.mel.islikelythesameasthefunctionind.ere.,sooncethefunctionis found,itissafetoassumeitsfunctionind.ere.isalsofound.thisgenehasorthologsin10closelyrelated Drosophilaspecies,soitoriginatedfromacommonancestorbetweenthese.Oncethereisareferencewithinone ofthese10,itsfunctioncanlikelybeappliedtoallbecausethissequenceismoderatelyconserved.orthologsare named(dspecies/name):dana\gf10942,dgri\gh15124,dmoj\gi13775,dper\gl25424,dpse\ga13080, Dsec\GM22428,Dsim\GD15018,Dvir\GJ13580,Dwil\GK10706andDyak\GE Exon 1 [flybase.org,genomebrowser scaffold_4784: ] Start End Stopcodon CG7139/NM_ ThisgenehastwomRNAproducts,ortwoisoforms,namedCG7139 PAandCG7139 PBinD.mel.Bothisoforms areconservedratherhighlyind.ere.,stillhaving4exonsinisoformaand2exonsinisoformb,bothrunningin theminusdirection.isoformbconsistsofthelasttwoexonsofisoforma,thusismissingthefirsttwoexonsfrom isoforma.thefunctionofthisgene,ineitherisoform,isnotknown.cg7139hasorthologsin10otherdrosophilid species,inwhichthey renamed(dspecies/name):dana\gf23494,dgri\gh16330,dmoj\gi11522,dper\gl25453, Dpse\GA20130,Dsec\GM22101,Dsim\GD12077,Dvir\GJ11777,Dwil\GK12132andDyak\GE22996.The coordinatesinfosmid16forisoformaare , , and ,withastopcodon 2

3 HarryQuedenfeld Genomics Dr.Moss at ThecoordinatesforisoformBare and withastopcodonat The lasttwoexonsinisoformaaretheexactsamepresentinisoformb;sincethefirstcodoninthethirdexonin isoformaisamethionine(astartcodon),isoformbbeginsatthesamelocation.thefirsttwoexonsofisoforma canbeexcludedtoyieldisoformb,thusthisgenecodesfortwomrnaandproteinproductsthatmayplay differingorsimilarrolesinacell.thisistheonlygeneinfosmid16thatdisplaystwomrnasforonegene.the proteinproductsyieldedbymyannotationofd.ere. sgenearesimilartothoseind.mel.blastpshowedanevalueof0.0andanidentityof85%withbothisoforms. CG7139 PA Exon Start End Stopcodon CG7139 PB Exon Start End Stopcodon CG7133/NM_ Thisgeneisasingleexongenetranscribedintheminusdirection.Thecodingregioncoordinatesinfosmid16is andthestopcodonisfrom Ithasasingleisoform,CG7133 PA.Thisgenehasitsgene ontologydefined.itsmolecularfunctionisdescribedasaproteinthatbindstounfoldedprotein.itfunctionsin heatshockprotein(hsp)binding.thusthisproteinisaheatshockproteinthatactsasachaperoneproteintofold unfoldedproteinsthatareeitherrecentlytranslatedorweredenaturedbyheat.thee valuefromablastp betweenthed.mel.cg7133proteinandthed.ere. sorthologousproteinis3e 81,andidentitysitsat55%.Sixtyfivepercentscoreaspositives,meaning10%ofaminoacidsthatdifferaresimilarinboth.Still,thislackof similarityissurprisingforsuchanimportantprotein.whenthed.mel.proteinisblastedagainstotherclosely relateddrosophilidspecies,noneofthemhavehighsimilarity,allhavingidentitiesaroundorlessthan50%.this eithershowsthatthisisanewmutationind.mel.thatcausesforafunctionalhsporitisafunctionalhspinall speciesbutdoesnotneedtoconserve50%ofitspeptidesequencetoremainfunctional.iftheformeristhecase, thenthatsupportsthatthisgeneisapseudogeneind.ere.therearenoothercopiesofasimilargeneind.ere., soidonotbelieveittobeapseudogene;rather,ibelievethatthisgeneisfunctionalinalloftherelatedspecies becauseonlyconservingacertainareaisnecessarytoretainitsfunction.infact,aboutthefirst65aminoacidsin CG7133 sproteinind.mel.showmoderatehomologytomanyotherrelatedspecies,whichsuggeststhisregionis moreselectedforthantheotherresiduesoftheprotein,whichsuggestsitmaybeessentialfortheprotein s interaction.thisgenehas4orthologsincloselyrelatedspecies,meaningitisanewergenethancg14561, CG7139andCG7130. Exon Start End Stopcodon CG7130/NM_

4 HarryQuedenfeld Genomics Dr.Moss Thisgeneisasingleexongenetranscribedintheminusdirection.Thecodingregioncoordinatesinfosmid16is andthestopcodonisfrom Ithasasingleisoform,CG7130 PA.Thisgenehasitsgene ontologydefined.itsmolecularfunctionisdescribedasaproteinthatbindstounfoldedprotein.itfunctionsin HSPbinding.ThusthisproteinisaHSPthatactsasachaperoneproteintofoldunfoldedproteinsthatareeither recentlytranslatedorweredenaturedbyheat.theblastpbetweenthed.mel.cg7130proteinandtheproteini predictedind.ere.yieldsane valueof1e 69anda92%aminoacididentity.SinceCG7130andCG7133arenot highlyparalogous,itisnotlikelythatcg7130cancompensateforalackoffunctionincg7133,whichfurther supportsthatcg7133isnotapseudogene(identity:32%).thispartialidentitycanbeaccountedforbythefact thattheyperformsimilarfunctions,buttheirstructurewouldbedifferentenoughthatonewouldmostlikelynot beabletocompensateforanother.thisgenehas10orthologs,whichmeansthishsphasbeenaroundlonger thancg7133.bothcg7130andcg7133areinthesameproteinfamily,hsp40 ( howevertheirdifferentstructureshavenotyetbeendetermined.atthistimethereisnotenoughinformationto determinewhethertheybindtothesamesubstrates,butihypothesizethattheydonotbecauseoftheirhighlack ofaminoacididentity. CG7130 Exon Start End Stopcodon RpLP0/NM_ ItsgeneontologyisdefinedashavingmolecularfunctionsinrepairingDNA,specificallyasaDNA (apurinicor apyrimidinicsite)lyaseactivity,whichisanenzymethatcutsoutnucleotidestobereplaced.insidethecellitis thoughttobeaconstituentofaribosome,anditsbiologicalfunctionsaretranslation,dnarepair,translational elongationandribosomebiogenesis.therewereprobablymistakesmadeinitsgodefinitionbecauseforitto repairdnaandbeapartofaribosomerequiredifferentstructures,forthesameproteintoperformsuch differentfunctionsisunlikely.itisatwo exongenecodingintheplusdirection.thecoordinatesfortheexons withinmyfosmidare: , ,withthestopat ;itcodesintheplusdirection.In D.mel.itisannotatedtohave3exons,howeverthefirstexonandaportionofthesecondarenottranslatedinto aminoacids,andarethusa3 untranslatedregion.the3 untranslatedregion(and1 st exon)isat , withthesecondexonstartingat11410butnotcodinguntil11450.ind.ere.however,thereisamissense mutationthatcausesforaprematurestartcodon,whichresultsinaprematurestopcodonbeingreadanda highlytruncatedanddissimilarprotein,thusthisusuallyuntranslated1 st exonmustnotbeanexonind.ere. becauserplp0ishighlyconservedamongdrosophilids.d.ere. srplp0contains2exons,thefirstisthesameas thecodingregionofthe2 nd exonind.mel.,andthe3 rd hasidenticalintron/exonborders.sincethe1 st exonind. mel.nolongerexistsasanexonind.ere.,theproteinsarethesamelengthandtheiridentityis98%,withanevalueof0.0.thusthisproteinishighlyconservedbetweenspeciesandperformsthesamefunctionind.ere.asin D.mel.RpLP0has10orthologousgenesindifferentDrosophilaspeciesincludingerecta.Theorthologousgenes arein(species/ortholog):dana\gf10946,dere\gg16244,dgri\gh14667,dmoj\gi13777\,dpse\ga20389, Dsec\GM22429,Dsim\GD15019,Dvir\GJ13582,Dwil\GK20443andDyak\RpLP0.Thetwomorecloselyrelated species,[r.1]dsecand[r.2]danaareshownbelow,bothofwhichalsoexcludethe3 untranslatedexoninorder toconservetheproteinsequence.figurer.3showsdmel srplp0nucleotidesequence,withafirstexonnoncodingandpartial2 nd non codingexon,indicatedbyblack,capitalletters.theseuntranslatedregionsmaybeade novomutationthatchangesthestartcodoninthefirstexon.thusdmelprobablyshowsanewinclusionofthis untranslatedexon,insteadofdereshowinganewexclusionbecauseotherspeciesaresimilartodere,notdmel. [Source:DecoratedFASTA,flybase.org] 4

[R.1] HarryQuedenfeld Genomics Dr.Moss [R.2] [R.

5 [R.1] HarryQuedenfeld Genomics Dr.Moss [R.2] [R.3] Exon Start End Stopcodon Query:D.mel.RpLP0mRNA,Sbjct:fosmid16 Thegreenboxbelowindicatesthemissensemutationandtheprematurestartcodonintheregionhomologousto the1 st exonind.mel.ifanexon,thisnormallyuntranslatedexonwouldbetranslatedind.ere.,thusitmustno 5

HarryQuedenfeld Genomics Dr.Moss longerbeanexonind.ere.becauseconservationofthisproteinisessential,with89%aminoacididentityor higheracrossd.sec.,d.ere.,d.ana.andd.yak.

6 HarryQuedenfeld Genomics Dr.Moss longerbeanexonind.ere.becauseconservationofthisproteinisessential,with89%aminoacididentityor higheracrossd.sec.,d.ere.,d.ana.andd.yak. [NCBIBlast2P] Ifthisprematurestartcodoniscodedfor,itwillresultinaproteinthatsharesnohomologywithRpLP0,asitis onlycodedforinthefirstexonandtherplp0geneind.mel.andotherspecieshasanuntranslatedfirstexon. Thisproteinwouldonlybe29aminoacidslongandwouldmostlikelybenonfunctional.InorderforD.ere.to survive,ihypothesizethatthisuntranslatedexonisexcludedintranscriptionofmrnaind.ere.thereisnoother proteinthatlookslikerplp0ind.ere.,sothisimportantgeneisnotapseudogenecopy. Sfp79B/ACG69555 NotonlyisSfp79BaveryshortproteininD.mel.ofonly35aminoacids,itistruncatedto4aminoacidsinD.ere., whicheliminatesanypossibilityofthesfp79bgenecodingforafunctionalproteinind.ere.basedonitslength,it mustbeapseudogene.asisseeninthefigurebelow,thereisamissensemutationthatcausesforapremature startcodon,whichafter9aminoacids,isfollowedbyastopcodon(taa).itwouldhavebeenasingleexongene intheplusdirection,iffunctional.sfp79bisaputativeseminalfluidproteinind.mel.becauseithasapredicted structuresimilartootherseminalfluidproteins(thusisnotconfirmed).blastpofthe4aminoacidsequence againstd.mel.yieldednosignificantresults.thisgeneisnotanywhereelseind.ere.,butsincetherearemany seminalfluidproteinsthatcancompensateforitsabsence,itdoesnotaffectfertility.itcouldalsobea pseudogeneind.mel.aswellbecauseitisshort(<100aminoacids)andhasnotbeencurated. 6

7 HarryQuedenfeld Genomics Dr.Moss 7 Query:D.mel. Sbjct:fosmid16(Tool:NCBI sblast2n) Msopa/NM_ Thisfeatureisprobablyapseudogenebecauseitisonly63aminoacidslong.TheD.mel.msopamayalsobea pseudogenebecauseitwasannotatedbylookingfororfsthatmightcodeforproteinsthatlooklikeacertain proteinfamily,andhasnotbeencurated.d.mel. smsopaproteinisonly83aminoacidslong.regardlessofits legitimacyasageneind.mel.,ind.ere.,msopahasa20aminoaciddeletioncomparedtod.mel.,issignificantly shorterandonlyhasa46%identitywithd.mel. s.thesethreefactspointtoitsbeingapseudogene.thisgeneis thoughttobeinvolvedinimmunityordefense,howevernothingisspecifiedbecausethisgenehasnotbeen researched.theproteinisnotknowntoexist,andat83aminoacidsitisunlikelytobeafunctionalgene.foritto existas63aminoacidsisevenmoreunlikely.msopadoesnotoccuranywhereelseinthed.ere.genome,the highestpercentidentityistheregioninmyfosmidwith46%sharedaminoacids.furthermore,msopaisnot conservedbetweenotherspecies(blastp68%identitywithdrosophilasechellia46%indrosophilayakuba,ncbi BlastP).Withsuchalackofconservationandsuchashortlength,itisprobablyapseudogene.Thisevidenceis strongerthanitsinferredexistenceduetopredictedstructuresimilaritybecausetheresearchersdidnotaccount foritslackofconservationinspecies.denovomutationsarehighlyunlikely.thereare4orthologstomsopain theotherdrosophilidspecies:dere\gg16245,dsec\gm22431,dsim\gd15020anddyak\ge22603,howeverin Dsimitis122aminoacidslong,almosttwiceaslongasDere ssupposedmsopaortholog.idonotagreethat GG16245isfunctionalinD.ere.orthatGM22431issimilarinD.sec.becauseitsproteinisonly67aminoacids long,giventhemedianpeptidelengthinalldrosophilidspeciesis373. [ GENSCAN Genscanpredicted6featuresformymaskedfosmid,threeofwhichweresingle exon.genscanpredictedsingleexongenesratherwell.thefirstexampleiscg14561,inwhichgenscanpredictedaregioninmyfosmidthat, whenblastedagainstmelanogaster,turnedouttobethemiddleofcg14561;thestartcodonwaslateandthe stopcodonwasearly,probablyinthewrongreadingframe.itpredictedthethirdexonofcg7139tobeadoubleexongene,whichisunderstandablebecauseitbeginswithastartcodon(asdoesisoformb)andthereisasplice junction(gt)rightbeforethestopcodon(tga),the T ofthestopcodonisthe T ofthe GT splicejunction. Forthethirdfeature,GenscanalsopredictedasingleexongeneinthemiddleofCG7133,butitonlytakesup aboutonethirdoftheactualgene.thisprobablyhappenedbecausegenscanreadinthewrongframe,causinga latestartcodonandaprematurestop.thefourthpredictedfeaturewassingle exonandturnedouttobe homologoustothecenterofcg7130,onlyslightlyshortonbothendsprobablyduetoreadingtheincorrectframe. Thefifthpredictedfeature,whenextractedfromfosmid16andblastedagainstD.mel.,ishighlyhomologousto therplp0gene,onlymissingthefirstexonandpredictingithastwo.genscandidpredictthisaccuratelyby overlookingthefirstexon,whichcontainsamutationforastartcodon.genscanmostlikelyskippedthisbecause theresultingproteinwouldbetooshort,thusitendeduppredictingrplp0correctly.thesixthfeaturepredicted atwo exongenethatspansfrom Whenthesebasesareextractedfromfosmid16,theyare homologouswithregionsaftermsopabuttherearenogenesind.melanogasterinanyofthehomologous regions.thegenscancoordinatesgivenmatchedupwithsplicejunctionsgtandag,butthereisastopcodonin thereadingframeandcutsthefirstpredictedexonshort,withnosplicejunctionnearby.duetothefactthat Genscanpredictedthefirstexonincorrectlyandtheexonsareverydistantfromeachother,thereisnotstrong

8 HarryQuedenfeld Genomics Dr.Moss evidenceforanewgenehereind.ere.thereare4intronspredictedtobebetweenthetwoexons,butthereare 10kbpbetweenthefirstexonandfirstintron,whichisnotlikelytooccurbecauseexonsandintronsareusually continuous.sincenoneofthegenscanpredictedgenesmatchedupwiththerealgenesperfectly,itcannotbe solelyreliedon;however,itprovestobeagoodstartingpoint.blastingtheregionofthepredictedfeatureagainst D.mel.willresultinhomologousregions,andGenomeViewwillshowifitispartofanygene,whichishelpful. CLUSTALanalysis RpLP0analysis AstheimagefromCLUSTALW2illustrates,RpLP0isahighlyconservedproteinbecauseithasanessentialfunction asaconstituentofaribosome.ribosomesarenecessaryineverycellallthetime,iftheymutatethecellwilldie.if anorganismhasamutationinrplp0,itwilllikelydie.thereare5mutationsthroughoutthe4organisms,butthey allresultinasimilaraminoacid.thismeanstheyhavethesamechargeand/orpolarity,sotheprotein sfunction wouldnotbealteredsignificantlybysuchamutation.key:fbpp :d.melanogasterrplp0,fbpp : D.sechelliaGM22429,FBpp :D.ananassaeGF10946,RpLP0 PA_peptide:D.ere. sorthologousproteinof RpLP0fromfosmid16,GG UpstreamRegions Thesealignmentsareperformedusingthe1,000bp5 upstreamfromrplp0ind.mel.anditsorthologsind.ere, D.sec.andD.ana.ThereisaconservedTATAboxinboththeD.ere.andD.mel.5 upstreamregions,shownby theredboxbelow.thetataboxisat inD.ere.and inD.mel.TATAboxusedhereisdefinedas anysequenceupstreamoftheinitiatorwith5of6nucleotidesconformingtotheconsensustataaa.(locations arebasedonthe1000bpextract5 ofrplp0oritsorthologs). 8

HarryQuedenfeld Genomics Dr.Moss ThefiguretotheleftillustratesthefrequencyofbaseswithinaTATAbox.[Source: http://jaspar.genereg.net/cgi bin/jaspar_db.pl] D.ana.alsohasaTATAbox[shownbelow]within20bpofD.

Isclearlytheleastsimilaramongthefour;however,D.melandD.ere.showahighersimilarityinthe 1,000bpbeforeRpLP0thantheothersdotothem.ThisisnotinagreementwithwhatisexpectedbecauseD.sec.

9 HarryQuedenfeld Genomics Dr.Moss ThefiguretotheleftillustratesthefrequencyofbaseswithinaTATAbox.[Source: bin/jaspar_db.pl] D.ana.alsohasaTATAbox[shownbelow]within20bpofD.ere. sand60ofd.mel. s,howeverd.sec.doesnot possessaqualitytatabox(ithasmorethanonedifferentbasefromtataaa)withinitsequivalent5 regionof DNA,thusitlikelydoesnothaveone.TheTATAboxlocationisat D.ana.Isclearlytheleastsimilaramongthefour;however,D.melandD.ere.showahighersimilarityinthe 1,000bpbeforeRpLP0thantheothersdotothem.ThisisnotinagreementwithwhatisexpectedbecauseD.sec. ismorecloselyrelatedtod.mel.thand.ere.is,howeverthesealignmentsattestthatthis5 upstreamregion havehigherhomologybetweend.mel.andd.ere.accordingtotataboxlocations.thefirst200basepairsofd. sec.andd.ana.notalignwithd.mel.andd.ere.becausethisregionwasnotconservedenoughtoalignbetween 4speciesperfectly,TheremighthavebeenaninsertioninD.secandD.ana.thatdoesnotalignwiththefirst250 basepairsoftheothertwospecies.d.ana.isclearlythemostdistantrelativeoftheother3species,asisvisible withthe showcolors optioninclustal. TheinitiatorCCATTGwasfoundinallfoursequencesinthealignment,indicatedbytheredboxbelow.D.ana.has amissensemutationthatleadsthectobereplacedwithag,butthisisstillaninitiationsequence. 9

HarryQuedenfeld Genomics Dr.Moss Adownstreampromoterelement(DPE)isany6nucleotidesequenceatexactly+28to+33with5of6nucleotides conformingtothedpefunctionalrangeseta/g/t C/G A/T C/T A/C/G C/T.

sequencegcttcawouldworkifthelasta wasat,sod.ana.alsoprobablydoesnothaveadpe28 353 oftheinitiator.[blackboxbelowindicates28 35bp aftertheinitiator,wheredpesarepossible].

10 HarryQuedenfeld Genomics Dr.Moss Adownstreampromoterelement(DPE)isany6nucleotidesequenceatexactly+28to+33with5of6nucleotides conformingtothedpefunctionalrangeseta/g/t C/G A/T C/T A/C/G C/T.Asseeninthefigurebelow, therearenodpeinanyofthespeciesbecausethereisacrichregionhere,whichcausesthethirdnucleotideto alwaysbeacandeliminatesthechanceforadperegion.thed.ana.sequencegcttcawouldworkifthelasta wasat,sod.ana.alsoprobablydoesnothaveadpe oftheinitiator.[blackboxbelowindicates28 35bp aftertheinitiator,wheredpesarepossible]. [Sourceforknownsequences: biology.ucsd.edu/labs/kadonaga/dcpd.html] Repeats [FromRepeatMasker] Mostoffosmid16ishighcomplexity,despitethelast20kbpbeingvacantofanygenesinD.mel.andD.ere.,only 6.31%isrepetitive,withnearly5%ofthatbeinginterspersedrepeats,ofwhichweremostlyDNAtransposons. GiventhattransposonsaccountforahigherpercentageoftheentireDrosophilagenomeonaverage,thelow percentageofdnatransposonsevidencesthatitisapossiblegene richarea.thereisonly0.15%ofmyfosmid composedofretroelements,whicharealsotypicallymuchmorecommon,whichshowsthisareahasbeen conserved.itisexpectedthat,anyorganismsthathadmanytransposonsorretroelementwouldhavemutations inthisareaandincreasetheirchanceofmutatingfatally.theorganismsthatliveddonothavemutationsinthese genescausedbytransposonelements,soweexpectlessrepeatsinagene richregion.however,thefirst20,000 bpoffosmid16actuallycontainnearlytwiceasmanyrepeats[fig2.2].thus,itisnotreliabletojudgewhethera regionisgenerichbytheproportionofrepeatsbecauseinthiscase,thegene richregionwithinalargerarea actuallyhasalmostalloftherepeats;2425of2526repeatswerefoundinthegene richregion.thisshowsthat transposonsandotherrepeatableelementsarepresentbutdonotcauseanyfatalmutations,andshedslightthat thecodingregionsmaynotbeexceptionallystable.[areasinyellowrefertospecificsmentioned] [2.1] Summary: ================================================== file name: RM2_Fosmid16.txt_ sequences: 1 10

11 total length: bp (40000 bp excl N/X-runs) GC level: % bases masked: 2526 bp ( 6.31 %) ================================================== number of length percentage elements* occupied of sequence Retroelements 1 60 bp 0.15 % SINEs: 0 0 bp 0.00 % Penelope 0 0 bp 0.00 % LINEs: 1 60 bp 0.15 % CRE/SLACS 0 0 bp 0.00 % L2/CR1/Rex 0 0 bp 0.00 % R1/LOA/Jockey 0 0 bp 0.00 % R2/R4/NeSL 0 0 bp 0.00 % RTE/Bov-B 0 0 bp 0.00 % L1/CIN4 0 0 bp 0.00 % LTR elements: 0 0 bp 0.00 % BEL/Pao 0 0 bp 0.00 % Ty1/Copia 0 0 bp 0.00 % Gypsy/DIRS1 0 0 bp 0.00 % Retroviral 0 0 bp 0.00 % HarryQuedenfeld Genomics Dr.Moss DNA transposons bp 4.75 % hobo-activator 0 0 bp 0.00 % Tc1-IS630-Pogo 0 0 bp 0.00 % En-Spm 0 0 bp 0.00 % MuDR-IS bp 0.00 % PiggyBac 0 0 bp 0.00 % Tourist/Harbinger 0 0 bp 0.00 % Other (Mirage, bp 1.73 % P-element, Transib) Rolling-circles 0 0 bp 0.00 % Unclassified: 0 0 bp 0.00 % Total interspersed repeats: 1959 bp 4.90 % Small RNA: 0 0 bp 0.00 % Satellites: 0 0 bp 0.00 % Simple repeats: bp 0.61 % Low complexity: bp 0.80 % [2.2] Summary: ================================================== file name: RM2sequpload_ sequences: 1 total length: bp (20000 bp excl N/X-runs) GC level: % bases masked: 2425 bp ( %) ================================================== 11

12 number of length percentage elements* occupied of sequence Retroelements 1 60 bp 0.30 % SINEs: 0 0 bp 0.00 % Penelope 0 0 bp 0.00 % LINEs: 1 60 bp 0.30 % CRE/SLACS 0 0 bp 0.00 % L2/CR1/Rex 0 0 bp 0.00 % R1/LOA/Jockey 0 0 bp 0.00 % R2/R4/NeSL 0 0 bp 0.00 % RTE/Bov-B 0 0 bp 0.00 % L1/CIN4 0 0 bp 0.00 % LTR elements: 0 0 bp 0.00 % BEL/Pao 0 0 bp 0.00 % Ty1/Copia 0 0 bp 0.00 % Gypsy/DIRS1 0 0 bp 0.00 % Retroviral 0 0 bp 0.00 % HarryQuedenfeld Genomics Dr.Moss DNA transposons bp 9.21 % hobo-activator 0 0 bp 0.00 % Tc1-IS630-Pogo 0 0 bp 0.00 % En-Spm 0 0 bp 0.00 % MuDR-IS bp 0.00 % PiggyBac 0 0 bp 0.00 % Tourist/Harbinger 0 0 bp 0.00 % Other (Mirage, bp 3.46 % P-element, Transib) Rolling-circles 0 0 bp 0.00 % Unclassified: 0 0 bp 0.00 % Total interspersed repeats: 1903 bp 9.52 % Small RNA: 0 0 bp 0.00 % Satellites: 0 0 bp 0.00 % Simple repeats: bp 1.04 % Low complexity: bp 1.56 % ================================================== Synteny Key:[Leftmost]verticalredlineatapproximately21735kbpinD.ere.isthestartoffosmid16.Fromthe3 isthe entirecodingregion,all3 aftermsopaisnon codingdnainbothd.mel.andd.ere. [1]OrthologybetweenDereScaffold_4784(top)andDmelChr3L(bottom) 12

13 HarryQuedenfeld Genomics Dr.Moss [flybase.orggbrowser] ThisshowshighsyntenybetweenDereandDmelbecauseallofthegenesinDereareinthesameorderasDmel s, codeinthesamedirectionandaresimilardistancesapart. [2]Fosmid16regioninD.ere. [UCSCGenomeBrowser: [3]Fosmid16regioninD.mel. 13

HarryQuedenfeld Genomics Dr.Moss [UCSCGenomeBrowser:http://genome.ucsc.edu] Theaboveimagesshowhomology[1]andrepeatelementsinDereandDmel[2,3].Fosmid16isonchromosome 3LinD.

14 HarryQuedenfeld Genomics Dr.Moss [UCSCGenomeBrowser: Theaboveimagesshowhomology[1]andrepeatelementsinDereandDmel[2,3].Fosmid16isonchromosome 3LinD.melanogasterandScaffold_4784inDere.Syntenyhasbeenpreservedbecausethegenesonfosmid16are allfromthesameregionofthed.melanogastergenome;thatis,theyareinthesameorder,spacedsimilarlyand onthesamechromosomeinbothd.ere.andd.mel.thusthereisnoevidenceofanychromosomalmutations suchasinversionsortranspositionsthathaveoccurredsincethesetwospeciessplit.therearerepeatspresentin Dere[2]betweenthefirstandsecondexon(minusdirection)thatarenotpresentinDmel(bluebox[2]),which suggestsatransposableelementinserteditselfbetweenthoseexons(thefirsttwoincg7139 PA).Thereisalso anotherinsertionofrepeatsinderebetweenrplp0andmsopthatisnotpresentindmel(redboxin[2]).the orangeboxin[3]showsrepeatsthatintervenecg1739andcg1733indmel,butcomeaftercg7133indere, whichevidencesminordnarearrangement;however,thisdoesnotalterthesyntenyofgenesinthisregion. 14

Classification of repetitive elements based on the analysis of protein domains. Pavel Neumann May 2018

Classification of repetitive elements based on the analysis of protein domains Pavel Neumann May 2018 A unified classification system for eukaryotic transposable elements (Wicker et al. 2007) Repbase classification