Oene, 93 (1990) 307-311

307

Elsevier GENE03~5

C h a r a c t e r i z a t i o n and nucleotide sequence of the gene encoding the human pyruvate dehydrogenase ~-subunit (Thiamine pyrophosphate-dependent enzyme; recombinant DNA; intron/exon junction; promoter; transcription start point)

Kiehiko Koike', Yoshishige Urata', Sachiko Matsuo b and Masahiko Koike" ° Department of Pathological Biochemistry, Atomic Disease Institute, Nagasaki Universe9, School of Medicine and b Animal Research Center. Institute of Tropical Medicine, Nagasaki University, Nagasaki 852 (Japan) Tel. (0958, ~7-2111 Received by Y. Sakaki: 21 February 1990 Revised: 8 April 1990 Accepted: 27 April 1990 .

SUMMARY

Genomic clones encompassing the entire gene (PDH~) encoding the human pyruvate dehydrogenase ~-subunit (PDH~t) have been isolated by screening a leukocyte genomic library in the cloning vector, ).EMBIA. The PDH~ gene spans 17 082 bp and is composed of eleven exons and ten introns. All intron/exon splice junctions follow the GT/AG rule. A total of seven Alu repeats were found in five introns. The entire nucleotide (nt) sequence of the PDHa gene has been determined and typical consensus promoter sequences in the 5'-flanking region were found. The results of primer extension analysis imply that the PDH~ gene transcription start point (tsp) is a thymine residue 124 bp upstream from the ATG start codon in exon 1. The structural organization and the tsp were compared with the recent report [Maragos et al., J. Biol. Chem. 264 (1989) 12294-12298]. Analysis ofthe PDH~ gene resolves existing discrepancies among four published sequences ofPDH~ cDNAs. A 93-bp sequence that was missing in our sequence of cultured foreskin fibroblast PDH~ cDNA [Koike et al., Proc. Natl. Acad. Sci. USA 85 (1988) 41-45] was identified in the gene as exon 6.

INTRODUCTION

Pyruvate dehydrogenase (PDH; EC 1.2.4.1) is a mitochondrial matrix enzyme encoded by a nuclear gene and is one of the component enzymes of the PDH multienzyme complex (Koike and Koike, 1976). It catalyzes the first step in the oxidative decarboxylation of pyruvate. Mammalian Correspondence to: Dr. K. Koike, Department of Pathological Biochemistry, Atomic Disease Institute, Nagasaki University School of Medicine, 12ban-4go, Sakamoto-machi, Nagasaki 852 (Japan) Tel. (0958)47-2111; Fax (0958)47-8514. Abbreviations: aa, amino acid(s); bp, base pair(s); nt, nucleotide(s); oligo, oligodeoxyribonucleotide; ORF, open reading frame; PDH, pyruvate dehydrogenase; PDH, gene (DNA) encoding PDH; PDHa, a-subunit ofPDH; PDHp,//-subunit ofPDH; pfu, plaque-forming unit(s); TPP, thiamine pyrophosphate; tsp, transcription start point(s). 0378-1119/90/$03.50 @ 1990ElsevierScience Publishers B.V.(Biomedical Division)

PDH is composed of two nonidentical subunits, • (PDH~; 41 kDa) and p(PDHp; 36 kDa) and has a tetrameric structure, ~ 2 (153 kDa). The activity of PDH is regulated by phosphorylation/dephosphorylation of three specific Ser residues in PDH~ (Yeaman et al., 1978). Recently we reported the aa sequences of human PDH~ and PDH~deduced from the nt sequences of human PDH~ and PDH~ cDNAs, which were isolated from a cultured foreskin fibroblast cDNA library (Koike et al., 1988). There are some discrepancies between the sequence of our PDH~t cDNA and the sequences of PDH~ cDNAs isolated from fetal liver (Dahl et al., 1987), hepatoma (DeMeirlalr et al., 1988) and liver (Ho et al., 1989) cDNA libraries. The major difference is the absence of a 93-bp sequence between nt 510 and 511 in our ORF. Partly to settle this controversy, we undertook the isolation of the human PDH~ gene. It has been reported that a genetic PDH deficiency in

308 GAATTCCCCQOATCTT11'UAOACAAA(~rAG'i'~AQACCATl'TACAQAAACCA(IATQ1~rCTQTCT'fTT GGCTCTQAQCATGCTGcrAATCTTCATCATCTAOTGTACTGAACQAGATGTACTCIAACQAGQGCTQCAQA GCTGCAQCACCGGCAGAGTA~CGCTCGGTAGGACGQGQCCTGCACAACCTCCCCGGTAGTCAOCAGAGC &&TCTAGGAAGGCTCCTTTCCCGCQGCGCCCTGGAGGCGGGGCCCCACCTTCCCACQCAGGCGCTATCA& GCCCCGCCTCCTCA¢CCGCCCQCGCTGG4GGTCGGAAAQAGCCCTCAGCCCCTCCCTCTCTGGCGCTGATA

-|Tt -;iO1 -lll -Ill

-II

Spl CCCA~TlSGGCAGCCTCAQGCCTTTArv1CGGGGGCGGGGCACCCCCTGGACGCCGTTCTGGTTGCCCGCG~ -~9 CAAT *! Sol CCQQ4~AGCQCATGACGTTAT~ACGA~G~CACGCCGCGGTQCGACTQAGGCGTGQCGT~T~T~G

4Z

TATA " . . . . . . . . . . GCACCTQAAGGAGACTTGGGGGCA~CCGCGTCGTGCCTCCTGGGTTQTGAGGAGTCGCCGCTGCCQCCAC 11

TGCC~GTGCTTCAS~GGAAGATGCTCGCCGCCGTCTCCCGCGTGCTGTCTGGCGCfTCTCAGAAGCCG~ i 1 ~ N





N

L

A

A

V

8



V

I.

8

Q

A

8

G

i

P|

Exon 1 I~AQAccIr~CCGGGCI~GCCGGATGGGGCGCGAGTGGGGCTG~GGGGCGAGGCAGGGCGGGCCAGGC CGGGCACCCAQAGCQGGGTOGAAGGCGCCAGGGGAGCO~GAGCCTTTACTTCQCCTCCGCGCCCTGCATT CCGTTCCTGGCCTCGGGAQAAC~;uGCA~ACCQATr~C~CCAAGGTCCGTOTGAACT~CCCCCTTCTCGA CACCCACCTCCCGCCCCCQGK]CCC~GClrGTGCGCCAGGCQAAGTCGGTGTGCTC&AGAGGTGCCTGTTGQ GTTACAGQACACGGAAAGGGTGGCCTCGGCTCCTTCGAGTCTCCA&TTGACCCCACTCATTTCGGATCTT CTAACTTAATTTCTCTTGACCGAGAGCTTTGTAATAGCGTAGA&TCTGGAGACAGGGTGGCTTCGTTCAA &CAGCACCCTCACCATTGACTA~CCTGTQACCTTOAGCAAGTTTTTAAACQTCCCGGGQACCCGTTTCC TAAAATGTTTGCTCQAAGTGOA~AATCTCTAAATGGAGATAAGAOTTATCTCTGAAATGTTATCGGTT ATTAAAATGTTATCAGTTAACTCTAAAATGGAGATAATAAOAGTCCCCACCTCTTGGGGTTGTCTTGAGO &TTCAACQAGTGACACGTGTGGAAACGATTCCAAATAGCAOCTGGCA~ATAATCOATAACATGTGTGTTG A&TAGTGTTATTTATTQAGTCTCCAGTTCGGTATACATTTCTTGAACCACCTGTGCTCAGTTCTGAGGCG GGTTCACAGAAGGTCAGCCTCTTCAQAAACAAACTTCCTCCTCTTCCCTCTCCCTCAACATCTGAGCTTT

||| 3== $9~ 46| I~| IO| IT| T4| l|| ll~ 9|~ 10=| log= IIII 1131 130= I~?|

TCTTGGCAGTGAGTTCAGGAGCGCCGAAGCAQAACTCAGAGQACGCTGCCCTCCCTCCCTTCCTACACAT CTTAGGGTACAGTAGCTAAAGCAAAGAGCAACGATGCTTGAGGGTGGGGQGTAGAGTTTAGCACTATTTC ATGGCGTCTAGCATTTAQAGGTCCTAACACCTQAGCTAGCAATTCTQACC~CGCTAGGCAG>QAGQTC GTGTTAATTGGTGTAACTGCAQQCCTCQGGATTCTGGTATTTCCCCCAGQACTTGATACCGCTCTACTTA GTACAGOCAAGAQATTGTCAAAAGGTAAAGAGGTATGCCCCTCTAGQAATCCTGTTGCCTAAAATAATGA CAAAACT~CCQGGTGCGGTGCTCAGQCCTGTAATCCCAGCATTTTGQGAOQCTGAQGCAGGTGGATCACC 1442

~ ...................... N u repeat t

TQACTAAQAATGQCATQAAAA~GCC&QACGCAQTGGCTCATGCCTGC~CCCAGTACTTTGGGAQGCC& 4101

j. ....................Alu repeat 2 .......................................... ~QGCAGGTGOATCACCTQAGGTCAGOAGTTGGAGACCAGCCTGGCCAACATGGTQAAACCGCATCTCTAC 41TI .................... o.........................o..................................................................................... TAAAAATATAAAAATTAGCCGGGCGTGGTGGTGGGCACCTGTAATCCCAGCTACTCGGQAGACTGAGACA 414| .................................................•..............................................................,................... GGAGAAATCACTTGAACCCGGGAAGCGGAGGTTGCAGTGAGCCGAG~CGCACCACTGCACTCCAGCCTG I I | | GGTGATAAAAGCAAAACTCCGTCTCAAA~CAAACAAACAAAAQAATGGC&TAAACAGACACAGCTCACAG 6 0 | | ATGATCTAGTCTCTTTAGCCACTAATTTCATTATATTCTCACTATAATTTCTTTOAAAACAAAGQATGSQ TTTOTTTTTT~CCCCTCTTTr.GCTGCTTQCCTTCA~ATGCQGGATAATCCTOTTT¢.T~OCCAAA~CAT aOATT©ATTTTgOAOGCCAAOOAAO~CAAAC&CAOTOCACAIGOTOQAAOAOAAlCCTATOAATATQT TG~SGCTTATT,AATTTCCATAACTTC,TTCTaAT,ACTa*TTATTATACTTTCCA~AATAGCTOA©,AT TAAA,,IT,CTa*TTTaTTTGTATA~TTTTOTCTTTTAA~SCAAOCAO*OTGCT.TAgCATCCCOT,AT

Exon 2

I||, |,|| I',| l=l' I¢='

TTTQCAAATGATGCTACATTTGAAATT&A~QTAAQAGTGTTTTACTTTGTT&ATAATTTTTTCACAGQTA F A N D A T P I ! ! |

||OI

CACTCTGATATACAGTTTTA¢CTTTAGAATAGAACATCTTGATGTTCATGATTAGTCATC~TTTCTTCT AAATGTCCAGGATCAGAAGTTCAGAGAAGCTTATTCAAAAGTTTGGAATQTA&TTCAQTQAAATAT~TGA ATAAOAAOAOTCTTAOTTOTTTC~TGAAQGTTCTTTCAACCTATAACTCAGTTGGCTTCTAGGGGCTTT CAGTQAAAATCATCTTAQAAAGATTTCCTTCC:CCCAAGCCCCATCTCATTGCACAGTGAGGTTTATQGAT TT*~OG,AC*OA~aCOATATOAAOCATT*¢TO,TaTaCTCCTTTr~AaTTTTTCAA~TTCAATATTATTT aC^ATaOAaTT,Q,TCTTAaAQTOGTCAACAQTaTTTaC~ATGTAGTATGT~QA~OATAATAACTACCTT kTTC,TTTC,O^AATaTOe¢CTrCACCa~CT~AkOAA~C¢CTCCTaTC~CAACAOT(~:TCACCA~SOA !K ~ ~ L p " L E E G P P V T T ¥ ~ T ~ B

llT| 114| ITI2 |T|| lU= ISll ln|

Exon 3

GGATGGGCTCAAATACTACAGGATGATGCAGACTGTAGGCCGAATGGAGTTGAAAGCAGATCAGCTQT&T l O l ~ D G L ~ T Y B N N G T ¥ R R N B L E A D Q L T AAACAaAAAATT~TTC~TaGTTTCTGTCACTTaTGTOATGGTCA~TOAGT~GTAGOTTTGTIGTGGAAC S|aZ K ¢ ~ ! I n a F c H L C p 0 ~!

......................................................................

TGAAQQfCAQAAGTTCGAGATCACICCTGG~CAACATGGTQAAACCCCGTCTCTACTAAAAATACAA&ATT

I|1|

...................................................................................................................................... AICCGQTCGTQOTGGCOQQCTCCTQTAATCCCAQCTACTCQQOAQQCTOAQOCGQOAQAATAGCOTQAAC i l l = ......................................................................................................................................

TGTGTTATTTAGGTACTQAAGTATGQCTTQTAOTT&TTGGGCTTTACCGTGCCATATGTATCAGAAQAGT I | O I TTQAGGCTGGTAATQTAATTTTCTTTTATTTATTTA.TTTTTTTQAIACAGTCTCTCTCTITCGCCCAQQT 8 | T | t ................., .................................., ........... TAOAGTACAQTOGTQATCTTGGCTCACTGCAGCCTCTGGTTAQAGTACAGTGTQATCTTGGCTCACTGCA li|41

CCGGQAGCGG~i~rTQCAGTGAGCGGAQATCGTGCCATTQCCATACGOCCTGQOCGAACAAOAGCAAGAC I I | | ............................. o.................................................................. , .....................................

Alu repeat 3 (Inverted) ............................................................. aCCTCTQTCCACTGGQCTCAAQCAATCCTCCCAC~TCAQCCTCCCQAGTATGTGGGACCACAGGTQCACA 1 4 i l .......................

TCCGTATTTTAAAAAAAAAAAAAAAAAAAAAAAAAAAA&GCGTTCCCTTTAQOATATCTGTGQGTAQAGG IT28

................................................................

..........................................................................

CCAACACACCCAGCTAATTTTTGT&TTTTTTGOAQATACGUGGTTTCACTATGTTGCCCAGQCTAGTCTC 8411 .....................................................................................................................................

~

QQTGTACCQQI'AQTTACQGGCTCAQAAACATCCTTCCTT~rAGQCACCTGATGTAQGTTTTCTTCTTCTTC i 7 9 |

. ....................................................................

TOCAAGTCAOOTTCATTQTTTCCTOTATCAGTTTGCAGOOT~CCCCCCCCCCCOCCACCTTACAGTAGQA | | 6 ~ AOAAAATTOAQTTCCAQATATQAAOTCACCTTTQAAAGTGCCCAGGTATCTTTCCACTTGOTGTQTAAAC i l S l

AAACTTCrGGQCTCAAGTGGTCCGCCCACCTTQGCCTCCCAAQQTQCTAQQATTACAGGCGTQAGCCAGTI l l |

TCTTCAQATAATTAQAAQTTTTCTOTOTCACTCAACTTQTCATOQACTAATTAQOAACCATTCCTQAAGC |O0~

GTQCCTCQCTQAAOCCAQTATTTTAQAATTAAAAAGTAQAATGCCAAAACCTGCTATQAAQCTTAGGCTA S i l l

......................................................................................................................................

,o,.

TOTTTQTATTTTQTAATTCTTCATACTTATQOATQTOTTTTTAGTTAACTATAAGTAACAAAATAQATCA ACQTTTTAQTTTTTTATATTATACATGTAAAAAOAOATTTTQCATATAAOCCTTTQACAAAAATOTTOAC AOTAAACAATAAQCAQTQOCTCAC¢CAAATTAQQCAQAQTTAQTQCACTAQACTCCTACCATQTOTQTOA TACTCCATQAAOQGAOQQAQAAQQQQAOOQAOAAOQOTAOQCAQCTOOTC~QATOGCTOTQACACAAOAT AATCCCCTTAACCTCCCAAOACOQTOTGTGTTTTTTCCTTTTTTATTCTCCCTQQTTTACTTTCQTTTTQ TTTOAUACAaQQTCTCTOTQTCACCCAOQCTOQAOTQCAQTAOCAOOACAQCTCACTOCAOCCTTAQCCT QCTQQQCTCAAOCOATCCTCCTOCCTTAOCCTCCTOAOTAQCTQOQAACACAQQCATQTQCCACCA¢CAQ ACCCAQCCAATTAAAAAAATTTTTTTTTTAQTAQAQACATQQTCTTQCTACOTTQCCCAQTCTQQTCTCC ATOTCCAQQCTCAAQCAOTCCTCCCACCTCQQCCTCCAAAQCTQOQATTACTCTCACTCTCTTAAAACCA QOCAOQTAQOQAOATTTATCTCAGQCTTAAAQATTQCCATTOTCTCATCAAAQAQTQTTTQOTQTOAAAC TTTQAAATOAATAT©AAOATTQTQTTTTTATTTTTQAATAAOQTTTATAQTTTTCATAQTTCTTATTTCA

1141 |lll Illl loll 1411 1411 Illl I181 ITOI ITTI Ilil

ATAACATTGCCAGTTTTCTGTACCTGTTCTTAQAGTTTTACTATTTTAAAACTTTOTaQ4= S i l l AQTATOATCOQCTGTAOTOTATATAATTTQOAQAQAAAQQATTAQTTTQTTTTTTQTTTTMGQaCTTAO I T l l QTCAAQOQTTAQAGTOAAATACOTACAAQOOQGAOCCAGQTAQAATAAATQAQTQ&AQAAOGC~AGGTATQ l | l ACAAAACAOAAAATQOTQAQAOQOAQTC&TGaTQAAC'IrQGCACCAQG&TGOQOTACCGAQAQQ&ATQCQA| | O I TOACTTOQTTCCAQCCAQTTOOTOQCATQTQQAAATCAQQQOTAATGTTTCCTQTTTTCCATQTCTAAQA I I T I OAAQQCOOAAQTCTQOATTTTCATQTQAAATTCCCAQTGTTTTAATQTTQAQATCTOATQTAQQOTTTTA TO41 TTTTAOQTCATCATACAQOAQAAAGOAAGOAAQTQQQACATQTQTGGOTTOC©AQTTTATTQCTTCTOGT T i l l TTQQQCCTTCQACTCTGTATTTTQOTGOAAAATAQCTAGTTTCTCTGGTTATTAATQACAOOTTOTACTA T i l l QCCQACATATTTCACTQTQQTCTAgOAAACQTTTTTATTTAGAAACATGTATCATATTGCCTCATAQTTT T i l l CTCQTTCCTCTAACACAOOAAQCTTQOTQTGTQGQCCTGQAQOCCQGGAT©AACCQQACAQACCATCTCAT | I | |• • & • C ~ ¥ g L , • ,, A, O . . . . I. . . . N . . . . .P. . . T D H L

Exon4

TQOAAQAAQATTOAATQCATTTAAAATQTTATTTTATTQTTTQCATTTCTQTATQQCTCCTTTTGTQAQA ||18 TCTTTACTAOCAATQTTTTQOCTTTATAAOTQOTAQOTAAQAATTTTAATTTACACTOTTAQAATCTQQA l l l l ATTTTTQAAAQGTI~TTCCTQTTTCACATQAATQQTTCCTATQTATTTAQQAAQTTAAAQTTTTACTTTT l o l l TTTTAATTAATTTTTTTTTTTAGGCTGQAATQCAGTGQCACAQTCATAOQTCACTGTAQCCTCAQGTGTG S | l a TQCCAOCATACCTOACTAATTTTTTAATATTTATTTTTGTAQAQATQAOAQTCTCATGTTQCCCAGQCTG ~ l l | QCTTTOAACTCCTQGCTTCAAQTQQTCCTCCCACCQTQQCCTCCCAAAQTQCTGGQQATTATAQGTGTQA QCCATCATQCCCQOCCTAGTTTTTATTTTTTAAAATTTGAGTQQQTTQTTCOTQQTCTCTGTCAQAQAQG AATCCCATTTAACAQAQAATCTTTTTATQQCTCTCQAQAOA&AATQA&TGQTAAACTTATCTTTTCACAA QCTCTCAQTCAGAAATOATACACACACAQTTCTQATAQQACTTTTAQCTTCTTTAAQTTTGTTCCTTTCA CTCATATCAQTQQTTCTTATTTTTQAQ&TACACAQTAATOAAQCC&TGQQAQAAAQTATCTAAQTAQCTT TCTQQCAQTCCTAATCTT'JrQCAQQCOCAAGTTACAQQCQCATGCCACAQQTACTOGQCOCCTTCTTGCTC TTATQTATAQ~ATTATCCTGCCTCATTGTTTCAACTCTAQQATTOAGAAAQAAQTTACCTTTTCTCTGTT ACTQTCQCOTQOCTQOTTTGQACTCCTG~CTTCCAA~AACTOCAQTTTCTQTAQTTGTATTTQUAAATTT ATTTCACAATACAATAAATTTCTQQCCCQACAAAATATTTATTAACTQCCA&Q&ATAACACATCTGTTTQ • TTC~rM**T*T*A~©*TTO*TTTaCTO~rTC~CCTTCTCTC,OC~rT,CTTCTTC©©~,TTCCTM**T TTCCTTC,CTTTTTCTO~O~T~C~TTAaTaa~¢TaTCTCTaCCTaT~AMT~CTO~CACTg,TT¢CT AOTATTTCAQTTQTTTTCCTCCAgCACTQTCATTOTCTQTQTTTQTTOG:TTTQTCCAMTMATQQTCTAT TQAOOOOTOM,OATMT~¢OTA4TT,QCTTTCTCCT4TTQ~CTTOT,C~CTCC,OOO4T,¢TTaOCAO~T¢ • OTCTTA*¢TCTTCTC,C¢,AOATCAOTCCAOTO¢TOOATT,OOT,,~T,TO,^C^C,TCAO,TOTOCT TTTTATGQAQAAATCATQTTGGTTTACA~GTCAQTQTGTQAGAATQTGQ~AGAAQQQAGCTAAAATAGTA TOATAATACTACTGQATAAATTTTQTGOT©TAACCTAAACCTTAGCCATTACATAQAAT&CTTTTQCTQT GAGCAQGTTTOCTCAQTTGTAAAACTQOAAAQQA&TCATTTCTCACCCCQC~CCTCCAAgI:TTACTCAAA ¢ATQACAGCCACCAAACATCA&QAOAACAQTGTTTCAQAQAA~ATTTCTACTQQQQC~T~AQQAIQAQCC TOTCCAAOATTT~CTaTTC~AMTTATAAMTTMTM**ACA,¢TQQCTC,MaTocCM6TTAOT~TTT**g TCT^O~O,aTQCT~aT,,TCTTTTCMTTT,TaTC,TT~OTQTCCCTM,&gT~TTT~TCATCAaT~SCOT T*C**TCT~MTTT,~A,T,TTCTC,TTCgT,TA¢,g~TACC~TTT~T,a~a~aT¢~OTTT~CCT,a

l|l| Sl~l |401 14T1 lie| llll Illl ITll IIII I.I lUl 401l 41oi 4I,| 4841 4~11 4ill 441, 4ill 4ill 4in ~mr~a~a~rr~A~T~mr'rTT~rTa~aT~T~TTTTaUCTTa~rC~Ta~T©Ta~rT~ 4T=l

TCACAGCCTACCQQQOTCACGQCTTTACTTTCAC~GQGQCCTTTCCGTCCOAGAAATTCTCGCAQAGCT T S l | I ~ A T | A H G F T F T | O L 8 V | E, I L A,, ~ L TA~A~QTTTGOTQTTGATTT&CAOA&AGGGQA&ATGAGTQGATTAAGTTTTTAAATATOTQTC&TTAAQA T i l l ..,,,.q QCTATTATOAGTTAATATTTGTT&AAAATTTTAAQTTTCTTTTTTTAA©CCTCTCTCCTTTGGTGCTC'ro T | | l GTAOTTOTGTTGTQCTCTTQAQTTAACTGACQ&TTTGTQAAGTTCTCTGGCCCCTCAGGTAAAAGTTTAA TlOI AACAQQTTGQTGCTATAAAATCACAOTAQQTTTQQTTATCATTCAAGCATGCCAGAAGAAQTCTAQCAGT TSTi CATAQAAAGTAAQTTCQGTTQAAGCsCTCCATGGTATGCAATGTAAATTCTAGAAATCTTCTTA&TATTQ TT41 CCCTTTTCTTTOTCCCCCQTQACTATTTQTTTGTTTTQGTGGTTTTTTTTTTTTTTTTTGAQACTGTGTC T S l l I TCA~TCCGTT GTCCMGOT~TaT QC,aTQOTaTO*TCA~aCTC*CTQC**CCTOACTCC~QQOTCAAQT T i l l .......................

A~Urepeat 4 (inverted)

...........................................................

a*TT~TC*TaCCTCC*C:~'CTa,UT,~"r~r,©~,T~,C©,CC,©,CCTaUCT,~TTTTTa T ~ l l ...................................................................................................................................... TM'rTTTTAQTMOWOM'~aOOTTTCM~eMTa'rraocc,Q~CTQ~TeTCCAACTCCTQACCTCAaaTQ,TCCl o l l ...................................................................................................................................... i'"¢-CT~¢TTQQCCT¢~:AAAOTOT' -"--""'--"--"'--'---Q~TO~QOTT*CAOgCOTQCAQCCgCA¢~r~ClTMTTTOTTTTTT """ |O,| .........................................................

TQAGACAqAGTCTCQCIrTTGTTQCCCAGGCTGGAGTGCAGTGGCQTGCCTCAGCCTCCCAAAATG~TAGGO l l l ATTACA~C~TGACGCCTGTQC~TCCTCCTCCTCCTCCTTTTTTTTTTTTTTTTTTOAGACAQ>T l l S l G G T ~ ; ~ ; ; ; ; ~ C ; ~ ; ~ ~

TCACTCTTT¢,¢CCAg~TOgAQT~iCTOgAgTa~aT ......................

Alia

repeat 6 (Inverted) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

cC~TCM*aC,MTTCTCC=aCCTCM~CCCT¢CTa,GT,OC~,Q~TT,T,OQTTTaCCC~,CC,CC, .....................................................................................................................................

ill| liT|

(Legend on pale 310)

309 CACCTGGCTAATTTCTGTATTTTTAOTAGAQACCAGQTTTTCACCATITTGGCCAGGCTGGTCITGAACT | 4 1 8 .........................................................................................

. .................... o.......... . . . .........

CTI GACCTCAGGTGATCCTACCCICTTCGGCCTCCCAAAATITIAGGATTACAGQCGTGCAGCIGITGCC

8111

............................................................................................ ,............................. o...........

CGGCCCICCTTQACTCTTGAACT&TGGTTGTCCTCTATATATCAGGGG&TT(KITICTAGGACC~TCQAGT i | i | ..... i

ATACAAAAATCCTCAAATACTCAAGTCCCAAAGTCAGCCTTCCAT&TCTTCIGGITTGCAICCTQAGAAT ATTCTATTTTCAATACATGTGTGGCTGAAAAAAAATCTGTG~ATAAGTITACCTGTGCAITICAAACCCT IITCAAIGATTQAATATATTTAGTGTACTAGTATAIQ&GAGGT~CTAAGATGTTTIIAACTIGCCAIAAA ACCCAQAAAAGTCCAG,GGIAICATCIGGATC~AACATCTIAAGGAAACIAAI11A~AIAGAGTAGGAAA AGCTGGAAAGGTTGAAGCACATGGAACTAGTIAAAIGACAA~AQAAACAT(~Ii~TGCCTGOAGGGACA GGTACTTAGACGACrGAAC~GGCCTCTQTGTTCTAATC~TTGAGCCTCAQAITACATAi~TG~TGCC~ TTT~TTTGC~TTIIAGAGTT~li~GIICIGCACATITGTATGTTCT~AITTCCAqGAC~AAAAC~A P n ! a

8133 el|t 1111

I1|| 19It 1003 1013

QAGACAGOOAAQ|GAQGS"T~AATQTAQTAACITTAUTAAGAGCTAAll~AQTAQAAT~A~HtOCA 1310t GQACCAAGACTACAOCT1GG~rACAATGGGCTG&GCAGTCTATGTCAiQ&CCCTAAOOCTI~QGAQlroccA 11113 GGCCCTGAGTGATGGGTA~CQCCACCTCI~CCCAATTCCI"GAAAAGAATACATGt~i~OC&GCCClq;OC 11843 TGGAGACAAGGGGAGTTGTAGAATG~AGTTGTAGA&TGTGIQACTITCCCAAACAGATA&CCCI-I~.ACAA 18V18 TTCTGAGTTAGGTGTTATTGTTTTG~CCATTTTGCAGACAAGGAAACllDAGCCATAGAGCAGTTAAAQGIr 18T88 ACACAGCTACTCTG~CTTGAGCCA~CTCGCCCATTrTAAClfl]CTGr.ACCAAACGAACCAAGGA t r a i t TCGTCAATGITICTGCACCCCAGTGTTCAQAQCCGICTCC~GQAA~AGIQIAQdUtlGAGCQAQC III111 CCAACIGTCCAQACTCCCAGCAGAGCCTl~GGGGTT1rGGGIAGCAGAGGllGIll~Sq3CCTQ~t ACIT11~T 111001 TACACACTAGCAAGGTCTGI"GAAGTAOGAGI"GCTGCCAGCCCCACAACACACCAIGAGAAAGGAQC&TI~k IIQII QATGGAAATCTGTCTAGCCAATAGCAGGAGGCTCTAQAACATGCTCAGAGCCTTTTT .CT.TTTTTCACAGQ 13111 GTCTCQCGCAQIGGCACACTCACA~CTCACTGCICCCT~GACCTICCAGAATCAGGT~TCCTCCC&AGT I l l O I

Alu

.......................

repeat 7 (inverted) .............................................................

AGCTGGACTACAGGCGCACGCCACCATGCC~GGCTAATTTTTGTATTTTTTOATAGAGAIlSGGGTITIl~

GGITiTGCTAAAGGGAAAGGAGGATCGATGCACATGIATGCCAAGAACTTCTACGGGGGCAATGGCATCG l i 4 t G C A K I K G G 8 N H N T A K N F T G 0 N G I

Exon 5

~GGQQAG~GC~?TAQICAAGGACGAGGATTGTGTGCTGCTTTAGATTTGGCCCTGGACTTTGTCTTG&A& 9118

AACCTTTCACAGCC¢CAGACAACTTTTCCTGAAGCTAGTACAGCCATGTGCTGCACAITQACGCT~TG~T 1118 CAATGTCGCATATATGATQTTQGACCCATAAGATTATAATGGAGCTGAAAAATTCCTCGTCGCCTAGTG& 11S3 TGTTGTAQTGGCACAACACATTACCTTTTCTACGTTTAGGTACACAAATATTTTGCCTACAGGATTCAGT 9 4 | t AGAGT~ACATOCTGTGCAGGOTTGTAQCCTAGQAGCAG~AGOTCTACTATACA@CCTAQGTGTQCAQTGG 1411 GCTGTACCATCTAGGTTCGTGCATTACAGTATOGTGTTCACATGACAAAATCGCCTAQTQATGCAATTCT IIII GAGAATATATCCCTGTTGTTAAGTQACGCGTGACTATTITGGGIQCTTGQTTT@CTTTTAAAOACCTAQT 9638 GCTTCATATCCTACCGTTTOAGAQATGAGTAOATTTGGATGOTQATTTATAATGTTTCCTTTTAQOTOTC 9101 TGCTQTTTTATAAGTAAGCAGGAACCTCTAGCAGTGGAGCCATAACCTTCCCCTTCCTAITTATATTTCA 9118 CTACATTAATTGCTTTATCTTQTCAACTTCATTTTGGGGTCCTTGTTCTCATCATCAGTTAQTGAATGAT 9848 GAAGAATTAACAGCACAAAATTATATCCGGACTGTTTCTTTT¢CTTTCTAATATATTAAGATTCTATTAT IIII GTGTTGTTTTTTTTTAAACCTAGGTTTTATTTTTCCTTTTGAAATGGAGTCTTGCTCAGCCGCCCAGGTG 9988

11118

CATGTTGTCCAGGCTAGTCTCGAACTCCTGGGCGCAAGTGCTCIGCCC&CCTCAGCI~CCCAAAGTGCTG 13348 GQAT~ACAGCATGAGCCACCATCCTQGCCTTCAAAGCCTTTTTQAI~CAAAACAAQCTQllrGCAGAT1~C

11411

Ao AAATo AQAACA.GATCAGTCAACCA 11411 ATA&GAAATTCGTGACAACTCAGAAGATCTOATAAGACTACACIGG&CQCTTAATAAAGGGCCTiCGTTI IIIII GAGGCCQTGGATTGCCGGCCTGTTCTTCCAGTCATCGTTCCTAACTAACTAACTGCTACCGGTTCTGTT~ IIIII TAG.GGGC¢CAT¢CTGATGGAGCTGCAGACTTACCGTTACCACGGACACAGTATGAGTGACCCTGGAQTC& 1 t l l 8 |G P I L N E L Q T ¥ R Y H G H I N 8 D P G i JJ

.

~

Exon 9

.......

site

TACGCTCATCKIQCAGTGTGGTTTCC&TAGGGGTGGGCTTTGAATGGTOTTACATGGCAAA&GCAACAC i l T I 3

AQQQCATTAATTAOTATCTCC~CTCATGQATTTCTOTGQTTCCTTTCTCGQTTOTCCTTAATOTTAG~ 1 0 4 7 8

- Site ATTTCAGTATTTGCTTTTGGAGCTAGATACCAGTTCA~TTC&TGTACGCAQTGTGTTGGGC~TGAAGTTA 1311t TCTGAAAGCAGTGCCTCCTAATAAGAAAGCTTTCTGAAAATGCCTACAGTGTATAGTGTGTGTGAQCACA 11008 AGCAGCAAITTTTAAAQAAAGAQTQTTATATACCTTAATTGTAOAGGQAAATTGTTACATAAAACAGGGC 139T8 TGATGGTAGTGTAGTATCTTGGGGGCATTTAGCTTITGTAGGCATTACCCCAAGTCTACACCTGGCATAC 14048 TCTACCTGAAGAGGATCAGAATTTGGAGATGTTCCTCTGTCTGGTATAGAGCT~AGGGTAACTGGGTGTT 14118 CAATCCGTTGAAATACATCAATCAAAAAAGC~AQGTTCCACTAQGAAGGCATQAATTTTACTTTTTllBC~ 14i83 TAGTAGCTCTGQCCTGTGATTCTGQAATCCCAGTTTTGACACAGTTGAT&AATAAGCCTTTGTAGAGTGO 1 4 8 | 1 ACTTCTAAGQAA&AATCATGTAGAGAGCACGATATGGAAAAATGCACTTTGTGTAAATCTAY3G11~AAA 14111 ATGQTAQAATCCTTTTAGTG~TACTTCAQATQATATAGGCATAAOATACATTGGTTT~GCTGGCTGTGCT 14311 TCTTTAGGQGGACTTAAGGGAQAAAGGCAAGGCACATGQATTTCCTGCTTGGCGGTCTGATGTCTCAAAU 14411 TCTAATTATCACCACACACACCATCTCTGCTGTCCCCACCCATGTAGTATACAQGAGCCCAAATGGGTGQ 14183 GACAAGTGACACTTCTTTAGAACCTTACATCTAAATCAAAOCAGCAAQC&AAAACTTQGCCCCTGTIGI~C 14|0| GOAATGCCAGGQAAQCCATQTGACTCACCAGTGTACGGTTTTCTAGAAAAGACAOAAGCAGTTATTACAG 14111 AATGTTAGGCTGCGTTCTGGTATTT~GAAAGTATAACACACTCTGCCAGCTATAGTGACATAAGCATCGT 14T43 ATGCCCCTTTGTTTCAQAAACACACTTCTGTATTTCACTCATTGGGACAATCCAACCCCTATACTAGTTT 14011 ¢TACACQCaTCCTTOICTCTACTGGAACTGCTCTTACTQATCGATTACTACTTTTCCCTCCCCATAO~v 14813

CCCCTGQQCQCTQQOATTGCTCTAQCCTGTAAQTATAATQOAAAAOATOAGGTCTQCCTQACTTTATATQ 10648 P L Q A G I A L A C E Y N Q K D | ¥ C L T ~ Y -•. . . . . . . . . . . . . . .

¢CQTACACQAGAAQAAATTCAGGAAGTAAQAAQTAAQAGTGACCCTATTATGCTTCI'CAAOOACAGO&TG 14118 R ~ R E B I Q | V | B | S D P | N L L E D | N

,oo,, .......................

Alu

repest 6 (inverted)

............................................................

CCGAGTAGCTGGQAATATAITTACQTGCCACCATGCCCAACCATTTTTTOTAT~TTTAGTAGAGACQQIG 10188 .............................................................................................................................. o....... TTTCACATCTTITCCAGGATIQTCTCGATCTGTGQACCT¢ITQATCTGCCCAAAUTGCTQQQATTACAGO I O i H CQTQCACCACGCCCQOCCAGQTTTTATTTTTTAACTCTTGAATGCAGAAATGTTAGTGTTACTQGTTAAA 10818 !

ATAOAACATAGTATTTATATATTACTTTAITICTTTATTOAAAATATCGIAGGTQGaATAAACAGAOAGA 10338 TAGGGTTGOAAGGAGAGTTTGTAGCAQCAGTGTAA~TTCTGTGTCAGATTCTGQCCAGQAGTOAAAATGC 10408

~t ~

Exon 6

a¢aATGaTocTacT^AC¢^~aTAATr^?GTCTCTTAACTTC¢CA^AA^C^a?CTT^TTTTC,AAaTCTTT I O e l |

O D a

A..A

N ,t

---

Exon

10 aTOAACAaCWATCTTac©,QTaTaa,Ao~,©T,A,~a~,©waTcwcTTaTTcwTaoTaaTTTowAaaTTu

V N 8

N L

A |

V |

8

L

I.n

||

^^T,TT?^©,oTTa**TTTc~,w,a**~:^acA~,~Tac~^~,aa?a***~,aCAAaTCC~,~aac~A

IO,38

aCT~;~A**aTTacc,ccccTOCacaTawc^a^aT~Ta~aTaaa~vccTCcA^accc,a***oTa,Ta~ I s l e t

aaanc,cAx~cc~a~a**aa,ac~cAccTT~acxc~,¢~c~aTac?~aaC~A,aaccc~a~aa~,aa

tom

,A^As~a~^~^c~c~c~a^TTTC~a,~aavaaACTaTa^AcCAC¢,X©,c~a~aac~**accc©~

t~ta8

Ycaa^~,^~^acu~©~a^aYcc~**©YC^alY~C~,~ac~c~©~aY~A©ca~a~,~cc©¢~a~cva~, aOCCAOCAcTcTa?oAAOCCcToT~cT~aAaoCTaATYCT?XaoTo©~oaTTcAc~cYaocT,Tccxa?o

io3,8 nioat

**TcYaTAYTCC~AAATCTTCVOA,TvAacA*c~aTTcasAc~ToWaTTA**aAoTT~¢~ccAaCA~C, aaTCCTCAOC^a**cTCT~oTTaaYACCT^^OcTacToYTCA~T©~*~*CCVVT~*CACYOT~A~T~A

II|~l

QQCCTGATAQATTTCATATTOATCTTTTTTCCAOTQTGTTCCTTACTQCTAICATQQCCCCAAAQAAACA AQTAQTAGTTOOTTTQTCACCTTCCTTAOTTOCAAQAQTATQATQCCTQCTACTTCTCCTCCACCACCCA CCCCQCTTTCCQTCACCACCCAAAQCTCQQTTTTAQAAQAGOAOOCCTTTCTQTOCTTTATOAAAQCTT? CTGTQCCAQICAQAQCAQCAQCTQTTAQAGATQATOAAQCCTOQAQAAAOAAQCCAAATQAAACCCCTTT TCGTAACTACTTCCAO.GGCCAQATATTCQAAGCTTACAACATGQCAQCTTTGTGOAAATTACCTTOTATT |• O Q ) F E A Y, ,,,N N A A L ,,, M E, L,J P ,, ¢ ~,I ,

allot ilITI

:::::,qo,^,v:o,~avaa~,o:a,aa^,ao,a^vva,aa~:a©:ac©©Ao.:acc,¢aac~a,vccv

imt

~

11841 11311 11381

Exon7

TTCATCTGTGAOA&TAATCGCTATGGAATQGGAACQTCTGTTGAGAGAGCGGCAGCCAGCACTQATTACT 11468 P I ¢ 8 N N R Y O H O ? 8 V B a A A A a T O V • te 3 . . . . . ACAAQAQAQQCQATTTCATTCCTGQGCTQAQAGTAAGQA~CCCTGTQGTGQGQ¢CGGGCCAACGGAAGQC I i | 1 ~

v

,

R o o ,

,

r

O L 8t

CAAQQCCAAQQQTATQTCCTTGT3CA~ACCCTTQACGATCTTAQAAACATTGQAQAGTTTCATTCTCATA 1 1 3 9 8 CAQOAQCAGQTCATQTQAAAGTAAAATGQTTTGQQOCAQTTQGAT~A,'TGTCGCCCCT¢CCCTGTTTAT I l i l l TACCAG~TGQATOOAATQGATATCCTGTQCOTCCQAQAOGCAACA,.~GTTTGCTQCTQCCTATTQTAGAT 11~38 | V D a N O_ | L C V R n A T R F A A A Y C R ~xor~O CTQQOAAQQTAAQQCTCTAACACO'rCTCCCGTAQTQACATTTATCTCTGQAAOTTCAAAQACTOCCTCCC ti808 8 Q Kt • ATGTGCCTGCTOAAOCTGTTAGTGGGTACCTGCTAATTGA~OTQCATGAGATGOAAGCAGAGTOAAQQAQ |i678 CAGQGCTCCTTTGOOTAQS~TGG?CTTOOTAOCTCAC¢?OCTOGQAAGCCTACOTTTCTCTCA?TTOQGQ I1348 OAAOTCCOfTCTGOTGCTTCCTCTQCTTTGOCCTOTCTTCATOACAACTOAT~TGCCTTTfCCTTAQOTT 1 3 o 1 8 AATTCTO?CCCTCCTCCCCACCCCCCATTAATCATOAQTC{CTTOAAOOAACAOATTGGOGATCCCCACA 1 8 O 3 8 GTGTCCAGCATAQAA?QTCATGTATACAA?AGOCATTTAATA?GTGTOTCTTATACAAATTAAACAOTAT asa3s GQTAOAAOCTCQTAATCT?AQTCTTOCTCAGTCCTAAGQATCTTTCCCTTTTCAOTOTATGQCAGTOAOO 1 8 | | | OAQATGOTAAOAGGOAQACTQQCCTAAAGAGGTTTCACTCCTTTGGCACTAGTATAGGCTTAGGAQGTTT 18|93 GGGTGTTCTCCCTAAATCAGTGTTCTACAQCCCCAGAAACAAGGTTTCTQAAOTTGGCACTTCAOC~CCA 18313 TACCATQTACOTTTAGQCAG~QATGTGAATCAGATGTGGCCAGCCAATGATGGAAATGTCACAAAGTOQA 18438

/• B

!

D ,iV 8i

V - B l

i

Exon 11

I

8

D,,..1.,...... A A q

F ~

T

18441

A D P

OAQCCACCTTTGGAAQAGCTGQQCTACCACATCTACI'CCAQCGACCCACCTTTTOAAOTTCQTQGTOCCAI l l l l B P P L E R L Q Y f l l Y 8 8 D P P P B V B Q A ATCAUTGGATCAAQTTTAAGTCAGTCAQTTAAGGGGAGQAQAAQGAQAQOTTATACCTTCAGGGQQCT&C Illll ~ ~ ~ , ! ~ P E, ~,, ~ 8 t e e CAGACAOTGTTCTCAACTTGGTTAAGGAGGAAGAAAACCCAQTCAATQAAATTCAATQAAATTCTTGQAA Illll ACTTCCATTAAQTGTGTAQATTGAGCAiiTAOTAATTGCATGCAQTTTGTACATTAGTOCATTAAAAQAT liVll ......

.

.

.

.

.

-.....

P~y (A),~dgnal

QAATTATTQAQTGCTTAAAQATT.ATTTTOACTTAAATAQTATACTTTQAACATACTCTAATTATOAAAO018808 . . . . . . . . . . .

1

AAOAACAATTCCTQTATQCCTOTTTCCCCTQCCCCCAAGCCCCCTTTAATTGGOAQOAAQACCATTATGG 18938 AAQOQOACCCATCACAQCAATTCTACCAACCATAOCACCCACCC¢OAOCAOCOCTOOTGCTOCAgCCTOT iiOOt TCOCQCTOACCATTTCTCTACAAGATACAATATTTATTATCAGOCAAQAGQACAOTTCCATfTTAAAATA lOOT| AGACTTTTGTAATCATTCCAATTTTGTAATCATTTCAAAGGCCACATAACTfAQTTTTCTCTACT?ACAC 11148 ATTCAGTATAAATATGAAGCTATTTTCTGTTCATATCAAACATTAACTACAAGGCACATTCQTATCAGTT 11813 TfGTGTTICTCAAATfQAAOTACCATACCAGTTCTQAQGCAQTGTCCCAGCTTCCATQT?TGTTAAATAC11|38 CCCTTGTTTGTTTCACCATTCCAGCAAGTOCTOAAGGOTOTACTTTTTTTOAOACAGOQTCOQOCTCfOTIS3St TGCCCAGGCTOOAGTGCAOTQOTOT{ATCATGOCTCACTOCAOCCTCCACACCTCCTQQQCTCAAQCAAT16483 CC?CCCACCTCAOCCTCCTGATAGCTGGOACTACAAGTOAA?TTCCTAAfATTCOBOOAQOTC&AA&CCA11408 AOGCTCACTGTTTTCACAATACACACAGTTCTATOTTfATAAATAACAGOfTTCAAAAQAAACTCAOQAC13|61 AOTAT?TAAAACAAOTTCTTAAACTATTAAT|OAACAATGUCATTTTTAAATATQTAAACACAQCOOAA? l U S t TC 11134 (L~ndo~l~

310 infants results in progressive brain and neurological disease with persistent lactic and pyruvic acidemia and growth retardation. The majority of these patients showed low levels of PDH activity and some ofthem showed lack ofone or both PDH subunits and appearance of abnormal protein bands by immunoblotting (Koike and Urata, 1989). Surprisingly little is known about the biochemical nature of human PDH, except for the recently published sequences of the PDHo: and PDH~ cDNAs. Molecular cloning and characterization of the human PDH gene should facilitate studies on the structures of the PDHo~and PDH~8cDNAs and genes and their expression. This knowledge may also provide information about the nature of genetic mutations leading to molecular diseases and aid in carrier detection, prenatal diagnosis and treatment. The aim of our study was to clone a human PDH~ gene and determine its entire nt sequence. Also, the recent report of the structural organization of a human PDH~ gene by Maragos et at. (1989) has been carefully examined.

EXPERIMENTALAND DISCUSSION

(a) Isolation of human PDH~ gene A genomic library of human leukocyte DNA constructed in ).EMBL4 was kindly provided by Drs. M. Takiguchi and M. Mort (Kumamoto University Medical School, Kumamoto, Japan). About 2 × 106 pfu of recombinant phage were screened with a nick-translated probe of human foreskin fibroblast PDH~ cDNA. Three positive clones ().GPDHAI3, 15 and 18) were isolated after tertiary screening. About 1.5 × 106 pfu of recombinant phage were rescreened with two 32p probes corresponding to the 5' end of the eDNA. One was a 57-mer synthetic oligo corre. sponding to the segment between nt I and 57 of the eDNA and the other a BamHl.Smal fragment of approx. 400 bp of the 5' end. One positive clone (AGPDHA37) which hybridized with these two probes was also isolated. Screening, hybridization, and washing procedures were carried out as described in Maniatis et al. (1982).

(b) Physical characterization of a human PDH~ gene The four individual DNA inserts were subcloned into pUC19 (pGPDHA13, 15, 18 and 37) and analyzed by restrictior~ analysis and mapping with a full-length PDHo~ eDNA probe. Two of the clones (pGPDHAI3 and 37) overlapped about 10kb, spanned about 17kb, and apparently contained the entire PDHu gene. The other clones (pGPDHAI5 and 18) were found to be included in the pGPDHA13 clone. When several synthetic internal oligos corresponding to several segments of the eDNA were used for hybridization, we found that one of them (pGPDHAI3) ended very close to the 5' end of the gene starting from the 3' end, whereas the other (pGPDHA37) contained the 5' end region. (C) Nucleotide sequence analysis of human PDH~ gene Fig. 1 shows the entire nt sequence of the PDHu gene. The PDH~ gene spans 17 082 bp and contains eleven exons and ten introns within the coding region. The eleven exons range in size from 60 bp (exon 2) to 366 bp (exon 11), and the ten introns range from 184 bp (intron 7) to 5221 bp (intron 1). Intron 1 is the longest. All intron/exon splice junctions follow the GT/AG rule. A missing 93-bp sequence was detected as exon 6, suggesting the possibility of deletion by abnormal splicing. Therefore, the nt and deduced aa sequences of PDHo~ eDNA previously reported (Koike et at., 1988) should be revised by insertion of the exon 6 sequence and by correction of minor differences. The aa sequence in mature pig PDH~ encoded by the 93-bp sequence was also confirmed by protein sequence analysis of PDH~ (unpublished data by Y. Urata). A total of seven Alu repeats was detected in five introns, Exon I consists of 181 bp and contains the start codon. It is interesting to note that the nt sequence encoding the Ser residue at phosphorylation site 2 (Yeaman et el., 1978) of the PDH~ straddles the splice site between exons 9 and 10. Maragos et at. (1989) have reported on the number, size, location of the exon and intron, and the nt sequence around the intron/exonjunction ofthe human PDH~gene. We have carefully compared it with our entire sequence and examined the discrepancies.

Fig, !. (on pages 308 and 309). Nucleotidesequence of the human PDH~8ene. The fragments of two clones (pGPDHAI3 and 37) were prepared by restriction digestion and by controlled digestionwith exonuclease BAL31 or exonuclease III + mung-beannuclease deletion. These fragments were subclonedinto pUCI9 and subjectedto the extensivesequenceanalysisafterstrand,denaturation,The nt sequencewas determinedby the dideoxynucleo. tide chain-terminationmethod. Nucleotides are numbered from the tsp (position + 1). The start codon is marked with three heavy dots. Exons are underlinedwithwavylinesand are numbered.The aa (standard one-lettersymbols)are printed under the codons. The sequencearound the phosphorylation sites in exons7, 9 and I0 are underlinedwithdashed lines.PhosphorylatedSer residues are designatedas Site I, Site 2 and Site3. Phosphorylation proceeded markedlyfaster at site 1 ti.an at sites 2 and 3. The Set codon of phosphorylationsite 2, at the end of exon 9 and the bef~inningof exon 10, is markedwith asterisks.A/u repeat sequences are underlinedwith dotted lines and are numbered. The sequence underlinedwith solid lines and their putative designationsare CAAT,CAATbox; TATA,TATA-iikebox; Spl, Spl-bindingsite; ATrAAA,poly(A)signal.The aa residues associated with TPP-binding sites in exons 6 and 7 are marked with open stars.

311

(d) Identification of the tsp for the human PDH~ gene The 5' end of the mRNA was determined by the primerextension method, using a 17-mer synthetic oligo primer complementary to nt 125-141 in exon 1 (Fig. 1). The primer apparently hybridized to poly(A) + RNAs from HeLa cells, human tissues (heart, liver and kidney) and pig heart and underwent extension by reverse transcriptase. The results of the primer extension analyses imply that the PDH~ gone tsp is located at the thymine residue (numbered + 1), 124 bp upstream from the Met start codon in exon 1 in contrast to adenine 105 bp from ATG as reported by Maragos et al. (1989).

ACKNOWLEDGEMENTS

The nt sequence for human PDH~ gene has been deposited in the DDBJ, EMBL and GenBank Nucleotide Sequence Databases under the accession number D90084. We thank Drs. M. Takiguchi and M. Mori for supplying the human leukocyte genomic library, Dr. L.J. Reed for helpful comments on the manuscript, Ms. S. Nakao for her helpful assistance and Miss M. Shiinoki for her assistance in preparation of the manuscript. This work was supported in part by a Grant-in-Aid for Scientific Research on Priority Area and for Scientific Research from the Ministry of Education, Science and Culture of Japan and by a grant from the Vitamin B Research Committee.

REFERENCES Dahl, H.-H.M., Hunt, S.M., Hutchison, W.H. and Brown, G.K.: The human pyruvate dehydrogenase complex. Isolation of eDNA clones for the EI~ subunit, sequence analysis, and characterization of the mRNA. J. Biol. Chem. 262 (1987) 7398-?403. DeMeirlair, L., Mackay, N., Lain, H.W.A.M. and Robinson, B.H.: Isolation of a full-length complementary DNA coding for human Elz subunit of the pyruvate dehydrogenase complex. J. Biol. Chem. 263 (1988) 1991-1995. Ho, L., We~der, I., Lin, T.-C., Thekkumkara, TJ. and Patel, M.S.: Characterization of cDNAs encoding human pyruvate dehydrogenase g subunit. Proc. Natl. Acad. Sci. USA 86 (1989) 5330-5334. Koike, M. and Koike, K.: Structure, assembly and function of mammalian ~-keto acid dehydrogenase complexes. Adv. Biophys. 9 (1975) 187-227. Koike, K. and Urata, Y.: Chronic acidemia due to a pyruvate dehydrogenase deficiency in pyruvate dehydrngenase complex, with abnormalities of the r, and ~subunits of'the enzyme. Ann. N.Y. Acad. Sci. 573 (1989) 450-452. Koike, K., Ohta, S., Urata, Y., Kagawa, Y. and Koike, M.: Cloning and sequencing ofcDNAs encoding 0¢and p subunits of human pyruvate dehydrogenase. Proc. Natl. Acad. Sci. USA 85 (1988) 41-.45. Maniatis, T., Fritsch, E.F. and Sambrook, J.: Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1982. Maragos, C., Hutchison, W.M., Hayasaka, K., Brown, G.K. and Dahl, H.-H.M.: Structural organization of the gone for the El~ subunit of the human pyruvate dehydrogenase complex. J. Biol. Chem. 264 (1989) 12294-12298. Yeaman, S.J.,Hutchison, E.T., Roche, T.E., Pettit,F.H., Brown, J.R., Reed, L.J.,Watson, D,C, and Dixon, G.H.: Sitesof phosphorylation on pyruvate dehydrngenase from bovine kidney and heart. Biochemistry 17 (19"18)2364-23"/0,

Characterization and nucleotide sequence of the gene encoding the human pyruvate dehydrogenase alpha-subunit.

Genomic clones encompassing the entire gene (PDH alpha) encoding the human pyruvate dehydrogenase alpha-subunit (PDH alpha) have been isolated by scre...
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