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Genomic Structure of Murine Mitochondrial DNA Polymerase- Gamma

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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
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  • Rare Allelic Forms of PRDM9 Associated with Childhood Leukemogenesis

    Rare Allelic Forms of PRDM9 Associated with Childhood Leukemogenesis

    Rare allelic forms of PRDM9 associated with childhood leukemogenesis Julie Hussin1,2, Daniel Sinnett2,3, Ferran Casals2, Youssef Idaghdour2, Vanessa Bruat2, Virginie Saillour2, Jasmine Healy2, Jean-Christophe Grenier2, Thibault de Malliard2, Stephan Busche4, Jean- François Spinella2, Mathieu Larivière2, Greg Gibson5, Anna Andersson6, Linda Holmfeldt6, Jing Ma6, Lei Wei6, Jinghui Zhang7, Gregor Andelfinger2,3, James R. Downing6, Charles G. Mullighan6, Philip Awadalla2,3* 1Departement of Biochemistry, Faculty of Medicine, University of Montreal, Canada 2Ste-Justine Hospital Research Centre, Montreal, Canada 3Department of Pediatrics, Faculty of Medicine, University of Montreal, Canada 4Department of Human Genetics, McGill University, Montreal, Canada 5Center for Integrative Genomics, School of Biology, Georgia Institute of Technology, Atlanta, Georgia, USA 6Department of Pathology, St. Jude Children's Research Hospital, Memphis, Tennessee, USA 7Department of Computational Biology and Bioinformatics, St. Jude Children's Research Hospital, Memphis, Tennessee, USA. *corresponding author : [email protected] SUPPLEMENTARY INFORMATION SUPPLEMENTARY METHODS 2 SUPPLEMENTARY RESULTS 5 SUPPLEMENTARY TABLES 11 Table S1. Coverage and SNPs statistics in the ALL quartet. 11 Table S2. Number of maternal and paternal recombination events per chromosome. 12 Table S3. PRDM9 alleles in the ALL quartet and 12 ALL trios based on read data and re-sequencing. 13 Table S4. PRDM9 alleles in an additional 10 ALL trios with B-ALL children based on read data. 15 Table S5. PRDM9 alleles in 76 French-Canadian individuals. 16 Table S6. B-ALL molecular subtypes for the 24 patients included in this study. 17 Table S7. PRDM9 alleles in 50 children from SJDALL cohort based on read data. 18 Table S8: Most frequent translocations and fusion genes in ALL.