Patchy Interspecific Sequence Similarities Efficiently Identify Positive cis-Regulatory Elements in the Sea Urchin

Developmental Biology

Lee Rowen¹  Eric H. Davidson¹  C.Titus Brown¹  Chiou-Hwa Yuh¹  Peter J.C. Clarke¹  Carolina B. Livi¹ 

[1] Division of Biology 156-29, California Institute of Technology, Pasadena, California, 91125

关键词: otx gene;    FamilyRelations;    cis-regulatory sequence;    computational genomics;    sea urchin embryo;   

DOI  :  10.1006/dbio.2002.0618

学科分类：生物科学（综合）

来源: Academic Press

【 摘 要 】

Wedemonstratethatinterspecificsequenceconservationcanprovideasystematicguidetotheidentificationoffunctionalcis-regulatoryelementswithinalargeexpanseofgenomicDNA.ThetestwascarriedoutontheotxgeneofStrongylocentrotuspurpuratus.Thisgeneplaysamajorroleinthegeneregulatorynetworkthatunderliesendomesodermspecificationintheembryo.Thecis-regulatoryorganizationoftheotxgeneisexpectedtobecomplex,becausethegenehasthreedifferentstartsites(X.Li,C.-K.Chuang,C.-A.Mao,L.M.Angerer,andW.H.Klein,1997,Dev.Biol.187,253–266),anditisexpressedinmanydifferentspatialdomainsoftheembryo.BACrecombinantscontainingtheotxgenewereisolatedfromStrongylocentrotuspurpuratusandLytechinusvariegatuslibraries,andtheorderedsequenceoftheseBACswasobtainedandannotated.SixtykilobasesofDNAflankingthegene,andincludedintheBACsequencefrombothspecies,werescannedcomputationallyforshortconservedsequenceelements.Forthispurpose,weusedanewlyconstructedsoftwarepackageassembledinourlaboratory,“FamilyRelations.”Thistoolallowsdetectionofsequencesimilaritiesaboveachosencriterionwithinslidingwindowssetat20–50bp.Seventeenpartiallyconservedregions,mostafewhundredbasepairslong,wereamplifiedfromtheS.purpuratusBACDNAbyPCR,insertedinanexpressionvectordrivingaCATreporter,andtestedforcis-regulatoryactivitybyinjectionintofertilizedS.purpuratuseggs.Theregulatoryactivityoftheseconstructswasassessedbywhole-mountinsituhybridization(WMISH)usingaprobeagainstCATmRNA.Ofthe17constructs,11constructsdisplayedspatiallyrestrictedregulatoryactivity,and6wereinactiveinthistest.Thedomainswithinwhichthecis-regulatoryconstructswereexpressedareapproximatelyconsistentwithresultsfromaWMISHstudyonotxexpressionintheembryo,inwhichweusedprobesspecificforthemRNAsgeneratedfromeachofthethreetranscriptionstartsites.Fourseparatecis-regulatoryelementsthatspecificallyproduceendomesodermalexpressionwereidentified,aswellasubiquitouslyactiveelements,andectoderm-specificelements.Weconfirmpredictionsfromotherworkwithrespecttotargetsitesforspecifictranscriptionfactorswithintheelementsthatexpressintheendoderm.

【 授权许可】

Unknown