University of Nebraska Medical Center DigitalCommons@UNMC Journal Articles: Biochemistry & Molecular Biology Biochemistry & Molecular Biology 10-2000 Genomic structure of murine mitochondrial DNA polymerase- gamma. Justin L. Mott University of Nebraska Medical Center, [email protected] Grace Denniger Saint Louis University Steve J. Zullo National Institute of Mental Health H. Peter Zassenhaus Saint Louis University Follow this and additional works at: https://digitalcommons.unmc.edu/com_bio_articles Part of the Medical Biochemistry Commons, and the Medical Molecular Biology Commons Recommended Citation Mott, Justin L.; Denniger, Grace; Zullo, Steve J.; and Zassenhaus, H. Peter, "Genomic structure of murine mitochondrial DNA polymerase-gamma." (2000). Journal Articles: Biochemistry & Molecular Biology. 3. https://digitalcommons.unmc.edu/com_bio_articles/3 This Article is brought to you for free and open access by the Biochemistry & Molecular Biology at DigitalCommons@UNMC. It has been accepted for inclusion in Journal Articles: Biochemistry & Molecular Biology by an authorized administrator of DigitalCommons@UNMC. For more information, please contact [email protected]. DNAANDCELLBIOLOGY Volume19,Number10,2000 MaryAnnLiebert,Inc. Pp.601–605 GenomicStructureofMurineMitochondrial DNAPolymerase-g JUSTINL.MOTT,1 GRACEDENNIGER,1 STEVEJ.ZULLO,2 andH.PETERZASSENHAUS1 ABSTRACT Wehave sequencedagenomiccloneof thegeneencodingthemousemitochondrialDNA polymerase.The geneconsistsof23exons,whichspanapproximately13.2kb,withexonsranginginsizefrom53to768bp. Allintron–exonboundariesconformtotheGT-AGrule.Bycomparisonwiththehumangenomicsequence, wefoundremarkableconservationofthegenestructure;theintron–exonbordersareinalmostidenticallo- cationsforthe22introns.The59 upstreamregioncontainsapproximately300bpofhomologybetweenthe mouseandhumansequencesthatpresumablycontainthepromoterelement.Thisregionlacksanyobvious TATAdomainandisrelativelyGCrich,consistentwiththehousekeepingfunctionofthemitochondrialDNA polymerase.Finally,withinthe59 flankingregion,bothmouseandhumangeneshavearegionof73bpwith highhomologytothetRNA-Arggene. INTRODUCTION thegenomicsequence(GenBankAccessionNo.AC005316)of the human Pol g gene (POLG) areknown, the genomic se- HE MITOCHONDRIAL GENOME is a circularDNA molecule quenceofthemouse Polg locusprovidesinformationaboutthe Tthatisreplicatedandmaintainedindependentlyofthenu- conservationof thegenomic structure.Furthermore,thepro- cleargenome. Multiplegenomes canexistwithin eachmito- motercan be tentativelyassigned by studying theconserved chondrion, andtherearehundreds tothousandsofmitochon- flankingDNA. driapercell.Theessentialrolethatmitochondriallyencoded proteinsplayinenergymetabolismunderscorestheimportance ofmaintainingthisextranucleargenome.Toreplicatethemi- tochondrial DNA (mtDNA), mitochondria have a dedicated MATERIALSANDMETHODS DNApolymerase,termedpolymerasegamma(Pol g;Lecrenier et al ., 1997). Like mostmitochondrial proteins,Pol g isen- Identification,isolation,andsequencing codedbythenucleusandimportedintomitochondria(Vooset al .,1999).Themurinegeneislocatedonchromosome7,and ColonyarraysobtainedfromResearchGeneticswereprobed thehumanhomologlocalizestoasyntenicregiononthelong for Polg using a probe generated from the cDNA. Positive armofchromosome15(Zullo etal .,1997).Toimproveourun- colonieswereobtained(ResearchGenetics)andsubclonedac- derstandingofthePol g gene,wehaveidentifiedandsequenced cordingtothemanufacturer’sinstructions.Southernblottingof a genomic clone containing themouse homolog (Polg). The DNAminiprepsrevealedthatBAC408-F6containedboth59 cDNAsequenceformousePolg waspreviouslyreportedby and39 terminiofthePolg cDNA.TheBACDNAwasmini- us(GenBankAccessionNo.U53584)*andusedtoidentifythe prepped accordingto themanufacturer’s instructionsand di- intron–exonjunctionsofthisgene. rectlysequencedusingdyeterminatortechnology(ABI)runon Asboth thecDNA(GenBankAccessionNo.002693) and anABIPrism310GeneticAnalyzerbycapillaryflow. 1DepartmentofMolecularMicrobiologyandImmunology,St.LouisUniversityHealthSciencesCenter,St.Louis,Missouri. 2LaboratoryofBiochemicalGenetics,NationalInstituteofMentalHealth,Bethesda,Maryland. *Sequencespreviouslydeterminedandreferencedinthispaperaremouse Polg cDNA(U53584),human POLG cDNA(002693;alsoU60325 andD84103),humanPOLGgenomic(AC005316),Drosophila tRNA-Arg(L09199),andhumantRNA-Arg(Z69590).Thenucleotidesequence datareportedinthisarticlehavebeensubmittedtoGenBankandhavebeenassignedtheaccessionnumbersAF268970,AF268971,AF268972, AF268973,AF268974,andAF268975. 601 602 MOTTETAL. Sequencecomparisons theexonsrangesfrom71%to95%,whileforintrons,therange isfrom55%to82%. Alignments and percenthomology weredetermined using theGCGprogram(GeneticsComputerGroup,Madison,WI), Putativepromoterregion whilerelatedsequencesweresearchedusingBLAST(Altschul etal .,1997).Allalignmentswerefurthersubjectedtoreview Onthebasisofhomologybetweenthemouseandhuman59 todetermineprecisejunctionsites.Togeneratesimultaneous upstreamregions,aputativepromoterregionhasbeenidenti- alignments,theClustalWprogramwasused(Thompson etal ., fied.Thereare297bpofhighlyhomologoussequenceimme- 1994). diatelyupstreamofthetranscriptionstartsite,with74%iden- tityfrommousetohuman.ThereisnoTATAbox,butaputative 9 RESULTS transcriptioninitiatorelement(5 GCAGACG;LoandSmale, 1996) overlapsthestartsite(basedonthefirstnucleotideof thecDNAsequence).ThereisanSp1-binding siteat 292to GenomicstructureofPolg 287,andthepromoter’soverallGCcontentisrelativelyhigh Approximately 14.8 kb of mouse genomic DNA werese- (63%).TheTATA-lesspromoterswithhighGCcontentoften quencedtodeterminethestructureof Polg .Thegenomicclone drivetheexpressionofhousekeepinggenes,consistentwitha wasintheformofabacterialartificialchromosome(BAC408- constitutiveneedformitochondrialDNAreplication.Promoter F6;ResearchGenetics)identifiedbycolonyblottingtocontain elementsknown toinfluenceexpressionofnucleargenesen- Polg (notshown).Theintron–exonjunctionswerededucedby coding mitochondrial proteins such as OXBOX-REBOX comparisonwiththecDNAsequenceusingtheGAPfunction (Chung etal .,1992),Mt3andMt4(Suzuki etal .,1991),and of the GCG program. Asillustratedin Figure 1, the geneis NRF-1 (EvansandScarpulla,1990) wereallabsentfromthe composedof23exonsand22introns.Alldonor–acceptorjunc- promoterregion.Thecorresponding regionofthehuman ge- tionsconformedtotheGT-AGrule(Table1). nomicsequenceisalsoTATA-lessandGCrich(64%)andhas Table1illustratesthestrikingconservationofintron–exon asingleSp1-bindingsite.Thereisnotaconsensusconcerning structurebetweenthemouseandhuman genes.Ofthe20in- the transcription start site for the human mRNA (compare ternalcoding exons,14areofidenticallengthwiththesame cDNAsequences002693, U60325, andD84103). Itisworth intronplacement.Lengthdifferenceswithintheremaining6in- mentioningthatthesequenceoftheputativetranscriptionini- ternalexons areminor, thelongestbeinga9-bp insertionin tiatorelementinthemousegeneis100%conservedinthehu- exon12ofthehumangene.Interestingly,besidesinsertionsof manandislocated29bpupstreamoftheearliestcDNAstart single-codonmultiples,oneortwonucleotidesareinserted(ex- sitereportedforthehuman(Lecrenier etal .,1997). ons4and15)intooneexon,onlytobedeletedfromthesub- Interestingly,73bpofabsolutelyconservedsequencedefine sequentexon.Exon2issignificantlyshorterinthemousegene theupstreamboundary of homology betweenmouse andhu- secondaryinparttotheabsenceofaCAG-repeatregion(Lecre- man genes (Fig. 2A). This high-homology area (the longest nier etal .,1997;Rovioetal .,1999).Mouseexonsrangefrom stretchof100% identitywithinthe ,14.8kbofgenomicse- 53to768bpinlength.Homologyfrommousetohumanwithin quence)isalsohighlyhomologous tothetRNA-Arggenesof FIG.1. Genomicstructureofmurine Polg.Exonsarenumberedandindicatedbyboxes,whileintronsandflankingsequences arerepresentedbyaline.Thetranscriptionstartsite(5 9)andthepoly(A)site(AAAA)areindicated,asarethetranslationstart (ATG)andstopcodons(TAA).BracketsmarkthebordersofhomologybetweenmurinePolg andhuman POLG.Thelocation ofatRNA-Arghomologisdenotedbythecloverleafsymbolnearthe59 end(seetextfordetails). GENOMICSTRUCTURE OFMURINEPOLc 603 TABLE 1. INTRON –EXON JUNCTIONS FOR MURINE POLG Exon Exon Intron Intron Exon identity Position size Splice Intron identity Splice size No. (%) incDNA (bp) donor no. (%) acceptor (bp) Mouse 1 79 1-124 124 GAGgtttn 1 70 tttagGAC 707 Human 1-123 123 .......c .......T 759 Mouse 2 86 125-892 768 CTGgtaag 2 61 ttcagGTA .1128 Human 124-941 818 ........ c....... 2817 Mouse 3 89 893-1088 196 CAGgtaag 3 62 tccagGAC .666 Human 942-1137 196 ........ ct....GT 970 Mouse 4 86 1089-1254 166 GCGgtgag 4 59 ctcagAAT 156 Human 1138-1305 168 ........ .c....TC 111 Mouse 5 83 1255-1403 149 CAGgtatg 5 56 gctagGAT 127 Human 1306-1452 147 ........ ..c....C 149 Mouse 6 89 1404-1483 80 GAGgtgag 6 82 cttagGTG 186 Human 1453-1532 80 ........ .ca..... 1106 Mouse 7 95 1484-1666 183 GAGgtagt 7 76 tgcagGTA 103 Human 1533-1715 183 .......c ..t..... 103 Mouse 8 89 1667-1815 149 AAGgtggg 8 67 cacagATC 136 Human 1716-1867 152 ........ ......C. 173 Mouse 9 76 1816-1942 127 TGGgtaag 9 59 aatagGTG 762 Human 1868-1994 127 .....g.. cg...A.. 926 Mouse 10 81 1943-2179 237 CAGgtaag 10 58 cacagAGC .513 Human 1995-2231 237 ........ .c...... 1034 Mouse 11 88 2180-2300 121 ACGgtgag 11 57 tctagGTA 195 Human 2232-2352 121 ........ ..c..... 205 Mouse 12 83 2301-2378 78 CTGgtgag 12 65 cacagCCT 286 Human 2353-2439 87 ........ .....A.. 303 Mouse 13 82 2379-2486 108 AAGgtatg 13 64 cacagGAC 444 Human 2440-2547 108 .....g.. tc.....T 501 Mouse 14 90 2487-2647