EDP Sciences httpwwwgsejournalorg or httpdxdoiorg101051gse2007024 ID: 135459
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Article published by EDP Sciences and available at http://www.gse-journal.org or http://dx.doi.org/10.1051/gse:2007024 Genet.Sel.Evol.39(2007)599607Availableonlineat: c INRA,EDPSciences,2007www.gse-journal.org DOI:10.1051 / gse:2007024 Originalarticle NNAT and DIRAS3 genesarepaternally expressedinpigs Huan-ChenC a ,Feng-WeiZhang a ,Chang-YanD a , Cao-DeJ b ,Yuan-ZhuX a ,Feng-EL a ,Ming-GangL a a KeyLaboratoryofAgriculturalAnimalGenetics,BreedingandReproductionofMinistryof EducationandKeyLaboratoryofSwineGeneticsandBreedingofMinistryofAgriculture, HuazhongAgricultureUniversity,Wuhan,P.R.China b DepartmentofBio-engineering,CollegeofAnimalSciences,SouthwestUniversity, Chongqing,400716,P.R.China (Received24December2006;accepted25April2007) Abstract Althoughexpressionandepigeneticdi erencesofimprintedgeneshavebeenexten- sivelycharacterisedinmanandthemouse,littleisknownonlivestockspecies.Inthisstudy,the polymorphism-basedapproachwasusedtodetecttheimprintingstatusof NNAT and DIRAS3 genesin veheterozygouspigs(basedonSNP)ofLargeWhiteandMeishanF 1 hybrids.The resultsshowthatbothgeneswerepaternallyexpressedinallthetestedtissues(heart,liver, spleen,lung,kidney,stomach,smallintestine,skeletalmuscle,fat,uterus,ovaryandpituitary). Inaddition,the NNAT genehadtwotranscriptsinalltestedtissues,whichisconsistentwithits counterpartinmanandcattle. pig / NNAT / DIRAS3 / imprinting / paternallyexpressed 1.INTRODUCTION Genomicimprintingisaparent-of-origin-dependentepigeneticmechanism, inwhichasubsetofautosomalgenesisexpressedfromonlyoneallele[15]. Imprintedgeneshaveimportantrolesintheregulationoffoetalgrowth,de- velopment,functionoftheplacentaandpostnatalbehaviour,inmammalsin particular[14].Atpresent,morethan120imprintedgeneshavebeenidenti ed inmanandmice,butonlytenimprintedgeneshavebeenidenti edinsheep, sevenincattleandthreeinpigs(http: // igc.otago.ac.nz / home.html).Therefore, itisofinteresttoidentifyotherimprintedgenesinpigsinordertoanalysethe conservationofgenomicimprintingamongdi erentspecies. Correspondingauthor:zfw2004790921521@126.com Imprintingofporcine NNAT and DIRAS3 genes 601 TableI. Primersequencesandproductsampli edfromtheporcine NNAT , DIRAS3 and GAPDH genes. AnnealingSize(bp)Accnoand GenePrimerSequence temperatureDNAcDNASNPposition NN1FACCCACCACCCTTGGAAC 595DQ666422 NNAT 58 C1758and NN1RGGCTTGATTGGCGCTGTC514noSNP NN2FCAGCACCGACAATGACGA DQ666422 NNAT 60 C645645 NN2RAATCTAGCCGGGGAGACA2135bp DIFCCCCATCAATA CCCAACGDQ666421 DIRAS3 55 C12271227 DIRTCTCTGC TCCCACCCTCA66bp DIIFCCCCATCAATA CCCAACGDQ666421 DIRAS3 56 C508508 DIIRCCTTCTCACTCACCTCCCnoSNP HouseFACCACAGTCCATGCCATCAC GAPDH 55 C780480 HouseRTCCACCACCCTGTTGCTGTA 2.3.PCRofDNAandcDNA Thehuman NNAT cDNAsequence(GenBank:NM_005386)and DI- RAS3 cDNAsequence(GenBank:NM_004675)wereusedtoiden- tifyporcineexpressedsequencetag s(EST)thr oughstandardBLAST (http: // www.ncbi.nlm.nih.gov / blast / )searchesintheEST-othersdatabase. PigESTsharingmorethan85%sequenceidentitywiththehumancDNA sequenceswereassembledintoEST-contigs.Theexon-intronstructuresof porcine NNAT and DIRAS3 geneswereestimatedaccordingtothestructureof thetwogenesinman.Allprimersweredesignedfromtheconsensussequences ofEST-contigs(Tab.I).ThePCRconditionswereasfollows:94 Cfor4min, 35cyclesof94 Cfor45s,annealingatoptimaltemperature(Tab.I),72 Cfor 1minanda nalextensionat72 Cfor7min.PrimerpairHouseF / HouseR, whichampli edthefragmentspanningintron8ofthe GAPDH genewasap- pliedtoexcludethepossibilityofDNAcontaminationduringallRT-PCRre- actions(Fig.1A). 604 H.-C.Cheng etal. Figure1. Expressionpatternsofporcine NNAT and DIRAS3 genesin12tissues analysedbyRT-PCR.Aistheampli cationwithprimerpairHouseF / HouseRofthe GAPDH genetoexcludetheDNAcontamination.Bistheexpressionpatternsampli- edwithprimerpairNN1F / NN1Rofthe NNAT gene,whichshowsthetwotranscripts ofthegene.Cistheexpressionpatternsampli edwithprimerpairDI1F / DI1Rofthe DIRAS3 gene. arepaternallyexpressedincattle.Ourresultswereconsistentwiththesere- portssincetheyindicatethatinalltissuestestedfromthe vetwo-monthold pigs,the NNAT genealsohastwotranscriptsandbothexpresspaternalalleles. Alternativesplicinghasrecentlyemergedasamajormechanismofgenerat- ingproteindiversityinhighereukaryotes[13].Thealternativesplicingofthe NNAT genemaybeusefultostudythefunctionofthe NNAT protein. Imprintingofporcine NNAT and DIRAS3 genes 605 Figure2. Imprintinganalysisofporcine NNAT (A,B,C,D,EandF)and DIRAS3 (G,H,I,J,KandL)genesrevealedbysequencing. ThearrowspointtoSNPsites.AandD,sequenceanalysisofgenomicDNAfromthehybridpigsshowsheterozygosity(C / T)at position2135 ofthe NNAT gene.BandE,sequenceanalysisofcDNAfromskele talmuscleshowsmonoallelicexpressionofalleles TandCatposition2135,respectively.CandF,sequenceanalysi sofmaternalgenomicDNAshowsallelesCandTatposition2135, respectively.GandJ,sequenceanalysisofgenomicDNAfromthehybridpigsshowsheterozygosity(G / T)atposition66of DIRAS3 gene.HandK,sequenceanalysisofcDNAfromskeletalmuscl eshowsmonoallelicexpressionofallelesTandGatposition66, respectively.IandL,sequenceanalysisofmaternalgenomicDNAshowsallelesGandTatposition66,respectively.Thematernal allelesarenotexpressedintheheterozygouspigs,soporcine NNAT and DIRAS3 genesarebothmaternallyimprintedandpreferentially expressthepaternalalleles.