Biochimica et Biophysica Acta, 1131 (1992) 83-90 83 © 1992 Elsevier Science Publishers B.V. All rights reserved 0167-4781/92/$05.00

BBAEXP 92376

Isolation and characterisation of the mouse pyruvate dehydrogenase E l a genes

James Fitzgerald, Wendy M. Hutchison and Hans-Henrik M. Dahl The Murdoch Institute for Research into Birth Defects, Royal Children's Hospital, Melbourne (Australia)

(Received 16 September 1991) (Revised manuscript 15 January 1992)

Key words: Pyruvate dehydrogenase gene; Nucleic acid sequence; (Mouse)

We have characterized two mouse genes that code for the E la subunit of pyruvate dehydrogenase (PDH), Pdha-1 and Pdha-2. The coding regions show a high degree of homology with each other and with the human PDH genes, PDHA1 and PDHA2. Conserved regions include mitochondrial import sequences, phosphorylation sites and a putative TPP binding site. The PDH genes have an analogous chromosomal arrangement to PGK genes in that two isoforms code for a functionally and structurally similar product. Pdha-1 codes for a somatic isoform and maps to the X-chromosome. Pdha-2 is located on an autosome, is intronless and only expressed in spermatogenic cells. Comparison of human and mouse PDH and PGK gene sequences shows that the somatic sequences are more conserved relative to the testis-specific isoforms, and that the mouse PDH E l a genes have experienced a faster rate of DNA change compared to their human counterparts.

Introduction

The pyruvate dehydrogenase complex (PDH) is cen- tral in aerobic energy metabolism [1]. The seven sub- units of this nuclear encoded enzyme complex are transported into the mitochondrion where they catal- yse the conversion of pyruvate to acetyl CoA via an oxidative decarboxylation step for entry into the citric acid cycle. The E1 enzyme is a heterote t ramer of two a and two /3 subunits designated P D H E l a and P D H El/3 [2].

The main mechanism of regulation of P D H activity is the phosphorylat ion/dephosphorylat ion of three ser- ine residues in the E l a subunit [3]. Most patients with P D H deficiency appear to have defects in the E l a subunit [4,5]. However, the clinical presentation of patients with PDH E l a deficiency is highly variable,

The Pdha-2 locus has been designated Pdhal by the Mouse Nomen- clature Committee. DNA sequence data from this article have been deposited with the GenBank Data Libraries (Accession No. M76727 (Pdha-1) and M76728 (Pdha-2)). The DNA sequence from the pro- moter region of the human testis-specific PDH Ela gene has also been deposited with the GenBank Data Libraries under the acces- sion number M86808.

Correspondence: H.-H.M. Dahl, The Murdoch Institute, Royal Chil- dren's Hospital, Flemington Rd., Parkville, Melbourne, Victoria, Australia 3052.

ranging from lethal lactic acidosis in the neonatal pe- riod to mild lactic acidemia with major brain abnormal- ities [6].

In situ hybridization of a human P D H E l a cDNA clone to human metaphase chromosomes revealed the presence of two loci [7]. PDHA1 was mapped to the X-chromosome (Xp22.1-22.2) and was found to be expressed in somatic tissues [7]. This gene contains 10 introns and spans approx. 17 kb [8]. The second locus, PDHA2, was localised to chromosome 4 (4q22-4q23) with expression limited to the testis [9]. This autoso- mal, testis-specific isoform completely lacks introns and possesses characteristics of a functional processed gene. Chromosome mapping in mouse has also re- vealed the presence of two E l a loci, one maps to the X-chromosome and the other to chromosome 19 [10].

The X-chromosome location of the somatic P D H E l a gene in humans, and the dependency of the brain on aerobic glucose oxidation are important factors when explaining the highly variable clinical presenta- tion of patients with P D H deficiency. Further under- standing of the clinical and biological effects of P D H E l a deficiency is hampered by the lack of a suitable animal model and problems with obtaining early hu- man fetal material. In addition, studies on PDH E l a gene regulation and expression in spermatogenic cells require the use of animal tissues. We have therefore initiated a study of P D H E l a expression in the mouse

84

TABLE I

Sequences of oligonucleotide primers"

Primer Sequence (5 ' - 3 ' )

Agtl0-1 Agtl0-2 Ag t l l -A Agtl 1-B mouse testis D mouse testis E mouse testis F mouse testis H mouse testis J mouse testis L

AGCAAGTTCAGCCTGGTTAA CTTATGAGTATTTCTTCCAG TCCTGGAGCCCGTCAGTATG ACTGGTAATGGTAGCGACCG TGCACATAAGTGGCTCAAGT GTTCTTGCCGTACATGTGCA TATAAAGATAGATTCTGAG GAGAGCTGGCTTI 'TGGACCA GACATAGCCTACTGTGCAGA GCCGGCCCTTCCTCCTTCT

and report here the isolation and characterisation of clones coding for the two mouse PDH E l a isoforms.

Materials and Methods

All DNA manipulations were carried out according to Sambrook et al. [11] unless otherwise stated. Oligo- nucleotide primer sequences are listed in Table I.

DNA probes Probe P D H A I : lc is a 1464 bp cDNA probe from

the human X-chromosome located PDHA1 locus. It contains 78 bp of the 5' untranslated region, the com- plete coding and 3' untranslated regions and part of the poly(A) tail [9].

ml PDH 10:1 is a 77 bp liver-specific EcoRV-SacI restriction fragment from the mouse liver eDNA clone ml E l a 10 (nucleotides 886-962 in Fig. 1).

mt PDH 1.2a is a 214 bp testis-specific PCR frag- ment (using mouse testis primers J and L) that codes for a sequence in the 5' untranslated region (nucleo- tides - 4 3 4 to -221 in Fig. 2).

Probes were labelled with a Random Primed DNA Labelling Kit (Boehringer-Mannheim).

All manipulations of DNA sequence data were per- formed using the MELBDBSYS, Walter and Eliza Hall Institute, Melbourne, Australia.

Isolation of mouse somatic PDH Ela cDNA (Pdha-1) Mouse liver E l a ZAP clone 2.1 was isolated from a

mouse liver cDNA library (Stratagene, catalogue No. 935302) in 3.ZAP. Approx. 130000 plaques (on Es- cherichia coli LE 392) were screened using the human PDHA1 : lc cDNA probe [9] and the filters washed to a stringency of 0.5 × SSC (at 65°C). A 3 kb cDNA clone was isolated and was subsequently found to con- tain the entire Pdha-1 coding region plus 100 bp of the 5' untranslated and 1.6 kb of 3' untranslated regions. The fragment was subcloned into the EcoRI site of M13mpl9 and sequenced using a Promega T7 sequenc-

ing kit. A partial clone (ml E l a 10) was also isolated from a Agtl0 library (Clonetech, ML 1017a), amplified in a PCR reaction using 3.gtl0 primers, subcloned into M13mpl0 and sequenced.

Isolation of mouse testis-specific PDH Ela cDNA (Pdha-2)

To detect the autosomal E l a isoform, an adult mouse testis Agtl0 cDNA library (Clonetech, ML1003a) was plated on E. coli LE 392 cells. Approx. 240000 plaques were probed with PDHA1 : lc and washed to a stringency of 0.5 × SSC (at 65°C). Two positive clones were detected and the inserts were isolated by using hgt l0 primers to PCR amplify the clones directly from the 3. supernatants. The PCR fragments were then gel purified, digested with EcoRI, ligated into M13mpl9 and sequenced. Both clones were found to be identical, containing 650 bp of coding region and 350 bp of 3' untranslated region. They were named mt E l a 2.4.

A 5' cDNA clone (rot E l a 3) was isolated by PCR amplification from a 3.gtll adult mouse testis cDNA library (kindly donated by Dr. E.M. Eddy) by amplify- ing with Agtll primer A and mouse testis primer E. Upon sequencing with mouse testis primer H the clone was found to contain 478 bp of coding region and 65 bp of the 5' noncoding region.

A 3. EMBL genomic library plated on E. coli NM538 cells was screened with a testis cDNA clone, mt E l a 2.4. Of 500000 plaques screened two positive clones were detected. The autosomal E l a gene was contained on a 3.2 kb XbaI fragment. This XbaI fragment was subcloned into M13mpl9 and named mt E l a 2.3. It was found to contain the complete coding region and flanking 3' and 5' untranslated regions.

Isolation and characterisation of Pdha-2 cDNA clones with poly(A) tails

Mouse testis cDNA clone 2.4 and 3 did not contain poly(A) tails. In order to define the 5' ends of PDH E l a mRNAs we characterized the 5' ends of addi- tional Pdha-2 cDNA clones. Screening of the adult mouse testis hg t l l cDNA library (Dr. E.M. Eddy) with PDHA1 : lc led to the isolation of an additional clone with a poly(A) tail. Asymmetric PCR was performed directly on this A cDNA clone using primers hg t l l -A and hg t l l -B (in excess) according to Saiki et al. [12] The PCR product was then sequenced with mouse testis primer D following the Sequenase protocol for single strand templates (USB).

Another cDNA containing a longer 5' untranslated region was identified using a Rapid Amplification of cDNA ends (RACE) protocol [13]. The PCR product was sequenced according to Bachmann et al. [14] with mouse testis primer F.

,4qG $;GA ATT TCA GGC CAA AAG GGG CAG AGA TGT AAC TAT GTG TAG AAT TGG GAT CAA CTG 60

2CA GGA GGA GTG CCT GGG TGC CGC GGC CGC CGC GTG AGT CTG CTG CGC TCC ATG AGG AAG 120/3

~t arg lys

ATG CTT GCC GCT GTA TCC CGC GTG TTG GCA GGC TCT GCG CAG AAG CCG C-CA AGC CGA GTG 188/23

met leu ala ala val set arg val leu ala gly set ala gln lys pro ala a ser arg val

CTG GTT OCT TCC CGT AAT TTT GCA AAT GAT GCT ACA TTT GAG ATT AAG AAA TGT GAC CTT 240/43 leu val ala set arg ash phe ala asn asp ala thr phe glu ile lys lys cys asp leu

CAT CGG CTA GAA GAG GGC CCC CCA GTC ACC ACA GTG CTC ACC AGA GAG GAT GGG CTC AAG 300/63 his arg leu glu glu gly pro pro val thr thr val leu thr arg qlu asp gly leu lys

'IAC TAC AGG ATG ATG CAG ACT GTG CGC CGG ATG GAG CTA AAG C.CG GAT CAG CTG TAT AAG 368/83 ryr tyr arg met met gln thr val arg arg ~t glu leu lys ala a asp gln leu tyr lys

CAG AAA ATC ATT CGT GGT ~TC TGT CAC TTG TGT GAT GGT CAG GAA GCC TGC TGC GTG GGC 420/103 QID lys ile ile arg gly phe cya his leu cys asp gly gln glu ala cys cys val gly

CTG GAG GCT GGC ATA AAC CCT ACG GAC CAC CTC ATC ACT C~C TAT CGA GCA CAT GGC TTC 480/123 !eu qlu ala qly ile ash pro thr asp his leu ile thr ala tyr arq ala his gly phe

ACC TTC ACT CGG GGC CTG CCT GTG CGA GCA ATT CTT GCA GAG CTA ACA GGA CGA AGA GGA 540/143 thz phe thr arg gly leu pro val arg ala ile leu ala glu leu thr gly arg arg gly

,5GT TGT GCT AAA GGG AAA GGC GGG TCA ATG CAC ATG TAC GCC AAG AAC TTC TAT GGA GGC 800/163 ql¥ cys ala lys gly lys gly gly ser ~t his ~t tyr &la lys asn phe tyr gly gly

AAC GGC ATC GTT GGA OCT CAG GTG CCC CTG GGA GCA GGA ATT GCC CTG GCC TC-C AAG TAC 660/183 asn gly ile val gly ala gln val pro leu gly ala gly ile ala leu ala Cys lya tyr

AAT GGA AAA GAT GAG GTC TGT TTG ACA TTA TAC GGC GAT GGT GCT GCT AAT CAG C~T CAG 720/203 a:~n gly lys asp glu val cys leu thr leu tyr gly asp gly ala ala ash gln gly gln

ATC TTT GAA OCT TAC AAT ATG GCA GCA CTG TGG AAA TTA CCT TGC ATT TTT ATC TGT GAG 780/223 lie pne glu ala tyr asn met ala ala leu trp lys leu pro cys ile phe ile Cys glu

AAC AAC CGC TAT GGC ATG GGG ACG TCT GTT GAG AGA GCA GCA GCC AGC ACG GAC TAC TAC 840/243 ~ s n ash arg tyr qly met gly thr ser val glu arg ala ala ala a set thr asp tyr tyr

AAA AGA GGA GAT TTT ATT CCT GGA CTC AGG GTA GAT GGA ATG GAT ATC TTG TGC GTC CGA 900/263 l>'s alg gly asp phe ile pro gly leu arg val asp gly met asp ile leu cys val arg

JAG GCA ACA AAG TTT GCG OCT GCC TAT TGC AGG TCT GGT AAG GGG CCC ATC CTG ATG GAG 960/283

~l,J ela thr lys phe ala ala ala tyr Cys arg set gly lys gly pro ile leu met glu

[TT( CA[~ ACF TAC CGC TAC CAT GGA CAC AGC ATG AGT GAC CCT GGA GTA AGC TAC CGC ACT 1020/303 ~t~: gin thL ~yr arg tyr his gly his set met set asp pro gly val mar tyr arg thr

?SA GAA GAA ATC CAG GAA GTA AGA AGT AAG AGT GAC CCT ATT ATG CTT CTC AAG GAT AGA !080/323 ~tq glu gLu ile gin glu val arg set lys set asp pro ile met leu leu lys asp arg

AT[; GTG AA:7 AGC AAT CTT GCA AGT GTT GAA GAA TTA AAG GAG ATT GAT GTG GAA GTG AGG 1140/343 tr,,t val ann sex ash leo ala Set val glu glu leu lys glu ile asp val glu val arg

AAA GAA ATC GAG GA7 OCT GCC CAG TTT GCC ACG GCT GAT CCT GAG CCC CCG TTG GAG GAA 1200/363 lys glu ile qlu asp ala ala gln phe ala thr ala asp pro glu pro pro leu glu glu

~TA GGC TAT CAC ATC TAC AGC AGT GAT CCT CCC TTT G~ GTG CGT GGT GCC AAC CAG TGG 1260/383 i eu qly tyr his ile tyr ser set asp pro pro pbe qlu val arg qly ala ash gln tip

A'r? AAG TTT AAG TCA GTC AGT TAA TGG GTG ACT GTT AGG TGG GTG OAT GGG TOT TCC TCA 1320

=le lys phe lys set val set OCH

FCC AGC ~TA AGG AAC TCT GTG CTC TCA ACT TGG ACA GOA AAT ACC CAG ACA ACA A.~A GGC 1380

7TO AAA ACA TGT A2~ 7TA AGG GAG AGT AAA ATT GCA TGC AGT TTG TAA AAA AAA AAA AAA 1440

Fig. 1. Nucleotide and amino acid sequence of the mouse liver PDH Ela (Pdha-1) cDNA sequence. A putative polyadenylation signal,

AAATTAA, is underlined.

Chromosome assignment Genomic DNA was isolated from male and female

mice according to Sambrook et al. [11] except the proteinase K step was omitted. The DNA was digested with restriction enzyme BglI, electrophoresed on a 0.7% agarose gel, blotted and probed with either ml

85

PDH 10" 1, a mouse liver-specific sequence, or mt PDH 1.2a, a mouse testis-specific sequence.

Results

Mouse liver E la isoform Sequence analysis of mouse liver E l a cDNA clones

10 and ZAP clone 2.1 revealed that the somatic iso- form of E l a has a coding region of 1170 nucleotides which encodes a protein of 390 amino acids (Fig. 1). ml ZAP clone 2.1 is a full length cDNA clone with an approx. 120 bp 5' untranslated region and a 1.6 kb 3' untranslated region terminating with a poly(A) tail. ml Elc~ clone 10 contains the 3' end of the coding region and a 143 nucleotide 3' untranslated region followed by a poly(A) tail (nucleotide 776-1446, Fig. 1).

Mouse testis-specific E la isoform Upon sequencing, the mouse testis E l a cDNA

clones 2.4 and 3 were found to be similar, but not identical to the liver cDNA clones. The Pdha-2 se- quence contains an open reading frame of 1173 nu- cleotides, thus encoding a 391 amino acid protein (Fig. 2).

Clone 2.4 spans nucleotides 744-1587 and clone 3 nucleotides 1-542 (Fig. 2). Clone 2.4 ends five nu- cleotides downstream from the second polyadenylation signal. Neither of these clones contain a poly(A) tail. The Agtll adult mouse testis library was rescreened and a positive Pdha-2 cDNA clone isolated, which was found to be polyadenylated at nucleotide 1347. The data from cDNA clone 2.4 suggested that a second polyadenylation signal may be used. To investigate this possibility the RACE protocol was employed. A cDNA clone was identified which was found to contain a poly(A) tail 17 nucleotides 3' to the polyadenylation signal at nucleotide 1578 (Fig. 2). Therefore, two types of cDNA clones were identified that differ in the length of 3' untranslated region. The shorter transcript includes a 3' untranslated region of 108 nucleotides with 10 bases separating an AU-rich sequence (AAUAAAU AAA) and the start of polyadenylation.

TABLE II

Amino acid homology for human mouse PDH EIo~ genes, human somatic (PDHA1), haman testis-specific (PDHA2), mouse somatic (Pdha-l) and mouse testis specific (Pdha-2)

Total number of Number of Amino acid Nucleotide amino acids amino acid homology (%) homology (%) compared changes

PDHA1/pdha- 1 390 7 98.2 88.6 PDHA1/PDHA2 387 53 86.3 85.5 PDHA1/pdha-2 390 88 77.4 76.5 PDHA2/pdha-! 387 52 86.5 81.4 pdha- 1/pdha-2 390 91 76.6 75.5 PDHA2/pdha-2 388 97 75.0 77.8

86

Two polyadenylation signals are encoded in this se- quence. The longer transcript contains 360 bases of 3' untranslated region and 17 bases separate the

TGT ATT GTA CAT ACT TTC AAT ~A TGT TAA GAT GTT GTT ATT TTT TTC TTT AAA AAC GAA 485

AAG GTA GAA GAA AGA ATG ATG TCA TTT TGC TCC CTC CCT CCT TCC CTC CTG CCT GCC CTT -425

CCT CCT TCT CCT CTT TTC CTC CTC TCC ACC GCC CCq CAT TTT GAG GCC AAA GAG ACC ACA 365

### #~# #J# ##~ ##J #

AAC TGA CTG AGA ATA GGA TTC AAG TCC GGC TCC TAC AI~A CCT CAA TGC CTA GTG TCT GAG 3C5

ATT ACA GGT ATG TGT GGC ACA GTT ACT GTC TGA GTT TAG AGG TGC TGG GAT GGA ACC C,,%A 245

@@@ @@@ @@@ @@@ @%@

AAQ CTC TGC ACA GTA GGC TAT GTC CCT ACC CTG CCA GGA CTT TTT TGA GTT GTA CAG ~A -185

AAA GAC ACT CA~ TTG CTG TGT GAG TGG CAA CCA TGA TGT TAA ATA GGA GGA AAG TGT GGG 125

AGA TCC TCC CTT TCC AGT TAG AAG CAT CAG AGG AGC AGG CCC CGT GGG CGT GGC TTC CAS 65

CTT GCT GAC GTA GGC AAC GCG TTT GCA TCC CGT TAT TGT TGC ATC AGA GGA GCT CGG CAG -5

+I CCA TCT TAA AAG CCA CTG AGT GAT CGT TGG GAG CCG AGC CGC TAC CGT TGT GCC TCG CGT 56

TTC TCC ATG AGG A~ ATG CTG ACC GCT GTG CTG TCT CAC GTA TTT TCG GQA ATG GTC CAA 116/18 met arg lys ~t leu thr ala val leu ~er his val phe set qly met val glrl

AAG CCA GCT CTC AGA GGA CTG CTG TCA TCT CTG AAG TTC TCC AAC GAC GCC ACC TGT GAC 176/38 lys pro ala leu arg gly leu leu set set leu lys phe set ash asp ala thr cys asp

ATT AAG A~ TGT GAC CTG TAC CGG CTG GAG GAS GGC CCA CCG ACC TCC ACC GTQ CTC ACC 236/!~ ile lys lys cys asp leu tyr arg leu glu glu gly pro pro thr set thr val le~ thr

CGA GCC GAG GCC CTC AAG TAC TAC CGG ACC ATG CAG GTA ATT CGO CGC ATG GAG TTG AAG 296;'78 arq ala glu ala leu lys tyr tyr arg thr met gln val ile arg arg met glu leu lyo

GCC GAC CAG CTG TAT AAG CAG AAA TTC ATC CGT GGT TTC TGT CAC CTG TOT GAT GGQ CAG 356,'98 ala asp gln leu tyr lys qln lys phe ile arg gly phe cys his leu Cys asp gly gln

GAA GCC TGC TGC GTG GGG CTG GAG GCA GGG ATA A.%T CCC ACG GAT CAC OTC ATC ACG TCC 416;'118 glu ala Cys Cys val gly leu glu ala qly ile ash pro thr asp his val ile thr set

TAC COG GCT CAT GGC TTC TGC TAC ACG CGA OGA CTG TCC GTG AAG TCC ATT CTC GCC GAG 476/138 tyr arg ala his gly phe Cy3 tyr thr arg gly leu set val lys set ile leu ala gltl

CTG ACT GGA CGC A~ GGA GGC TGT GCT A~ GGC AJ~G GGA GGC TCC ATG CAC ATG TAC GGC 536/15@ leu thr gly arg lys gly gly cys ala lys gly lys gly gly set ~t his met tyr gly

A~G AAC TTC TAC GGT C, GC AAT GGC ATT GTT GGG C43C CAG GTA CCC CTG GGA GCT GGT GTG 5@6/178 lys ash phe tyr gly gly ash gly ile val gly alaa gln val pro leu gly ala gly val

GCT TTT GCC TGT A~ TAC CTG AAG AAT GGT CAG GTC TGC TTG C4CT TTG TAC GC~ GAT GGT 656/]9@ ala phe ala cys lys tyr leu lys ash qly gln val Cys leu sls a leu tyr gly asp gly

GCG GCT AAC CAA GGG CAG GTA TTC GAA C~CA TAC AAT ATG TCA GCC TTG TGG KAA TTA CCC 716/218 als ala ash gln gly gln val phe glu ala tyr ash met set alaa leu trp ly3 leu pro

TGT GTT TTC ATC TGT GAG AAT AAC CTC T AT C~A ATG GGA ACC TCC AAC GAG AGA TCA GCA 776/238 cys qal phe ile cys glu ash ash leu tyr gly ~t gly thr set ash glu arg set als

GCC AGT ACT GAT TAC CAC AAG ~ GGT TTT ATT ATC CCC C~A CTG AGG GTG AAT GQG ATG @36/258 ala set thr asp tyr his lys lys gly phe ile ile pro gly leu arg val ash gly ~t

........................... I~ .... a~A a~ ............. ~ ............ asp ile leu cy3 val arg glu al& thr s phe a a a a asp his Cy3 srg set gly lys

GGG CCC ATT GTG ATG GAG CTG CAG ACC TAC CGT TAT CAT GGA CAC AGT ATG AGC GAC CCA 956/298 gly pro ile val met glu leu gln thr tyr arg tyr his gly his set ~t set asp pro

GGG ATC AGT TAT CGT TCA CGA GAA GA A G TT C AT AAC GTG AGA AGT AAG AGT GAT CCT ATA 1016/3] gly ile ser tyr arg set srg glu glu val his ash val srg set lys set asp pro ile

ATG CTG CTC CGA GAG AGA ATT ATC AGC AAC AAC CTC AGC AAT ATT G~ GAA TTG AAA GAA 1076/33 met leu leu arg glu srg ile ile set ash ash leu set ash ile glu glu leu lys ql~

ATT GAT GCA GAT GTG ~J~G AAA GAG GTG GAG GAC GCA GCT CAG TTT GCT ACG ACT GAT CCA 1!36/35 ~]e asp ala asp val lys lys glu val glu asp ala ala gln phe ala thr thr asp pro

GAA CCA GCT GTG G AA GAT ATA GCC AAT TAC CTC TAC CAC C AA GAT CCA C CT TTT GAA GTC ]196/37 glu pzo ala val glu asp ile ala ash tyr leu [yr his gln esp pro pro phe glu val

CGr GGT GCA CAT AAG TGG CTC AAG TAT AAG TCC CAC AGT TAG ATA SAT ~TT ACC TAT ACA 1256 erg g!y als his lys trp leu lys tyr lys ser his set AMB

T2T GZT AAA TTT TTT TTC AGT GGG ACA TTT ATG GTG TAC TC~ AGG A~ CTT CAA CTT TGT 1316

TAA GGA GGA ATA AAT AAA ACG ACA TTG CAG ACA AAA GTC TTA TAA ACC TTT ATA AAG ATA 1376

~AT rCC TGA GTT ATY ~O QAG ATT AGA AGA TAT ~ TTT GTT T~ ASG AGA FQT TCC ACT 143t

TTC TGT TTT AAC ATT A~ AGC ATT GTG TTG CAT ACT ACT ATG AAT ATC TTT TAG ACT ATT 1496

TCA AAT TTA TAA AAT TAT A~T AGA AAA AAC GGG TTA AAT TCC CCA ATT TGG CAT AGT AGT 1566

- %@@ @@@ @@@ ~@~ @@@

TT C ATT TGQ TTT TAG TTT TGA AAT AAA TAC TTT TAT TTA ~ G~ GTA GAT TTT TGA TTT ]6 ]6

TTT CTT CTT CTT TGA TGA GTT TGT C~ A~ TTA ~ GTG AGG AGQ ATQ ~TT TCA ATT CT~ 1696

TCC CCG CCC CCC CCT CAC CAA ATA CCT GAT TAT GTT CCC CTT TCC CCC TCC ATG q ? AA7 ] ')*

CTC TGA CCC AGG TTC CTC CCT CCC TCT GTC CCC TGT GGT TGA TTT TTC CTC CTA AGT AG] l~l~

PDHAI

pdha-i

PDHA2

pdha-2

PDHAI

pdha-I

PDHA2

pdha-2

PDHAI

pdha-i

PDHA2

pdha-2

PDHAI

pdha-i

PDHA2

pdha-2

?DHAI

pdha-i

PDHA2

pdha-2

PDHAI

pdha-]

PDHA2

pdha-2

PDHAI

pdha-i

PDHA2

pdha-2

PDHA]

pdha-]

?DHA2

pdha-2

1 50

kLRI~V SRg"LSGASQKPASR~FLVASRNFANDATFEIKKCDLHRLEEGP

~L*KK~AAV SRVLAGSAQKPASRVLVAS~FANDATFEIKKCDLHRLEEGP

MLAAFISRVLRRVAQKS~d~R~VASRNSSNDATFEIKKCDLYLLEEGP

~KMLTAVLSHVFSGMVQKPALRGLLSSLKFSNDATCDIKKCD LYRLEEGP

51 I00

PVTTVLTREDGLKYYRMMQTVRRMELKADQLYKQKIIRGFCHLCDGQEAC

PVTTVLTREDGLKYYRM}4QTVRRMELKADQLYKQKIIRGFCHLCDGQEAC

PVTTVLTRAEGLKYYRMMLTVRRMELKADQLYKQKFIRGFCHLCDGQEAC

PTSTVLTRAEALKYYRTMQVIRRMELKADQLYKQKFIRGFCHLCDGQEAC

I01 150

CVGLEAGINPTDHLITAYRAHGFTFTRGLSVREILAELTGRKGGCAKGKG

CVGLEAGINPTDHLITAYRAHGFTFTRGLPVRAILAELTGRRGGCAKGKG

CVGLEAGINPSDHVITSYRAHGVCYTRGLSVRSILAELTGRRGGCAKGKG

CVGLEAGINPTDHVITSYRAHGFCYTRGLSVKSILAELTGRKGGCAKGKG

151 200

GSMHMYAKNFYGGNGIVGAQVPLGAGIALACKYNGKDEVCLTLYGDGAAN

GSMHMYAKNFYGGNGIVGAQVPLGAGIALACKYNGKDEVCLTLYGDGAAN

GSMHMYTKNFYGGNGIVGAQGPLGAGIALACKYKGNDEICLTLYGDGAAN

GSMHMYGKNFYGGNGIVGAQVPLGAGVAFACKYLKNGQVCLALYGDGAAN

201 ** P 3 250

QGQIFEAYNMAALWKLPCIFICENNRYGMGTSVERAAASTDYYKRGDFIP

QGQIFEAYNMAALWKLPCIFICENNRYGMGTSVERAAASTDYYKRGDFIP

QGQIAEAFNMAALWKLPCVFICENNLYGMGTSTERAAASPDYYKRGNFIP

QGQVFEAYNMSALWKIPCVFICENNLYGMGTSNERSAASTDYHKKGFIIP

251 P1 P2

GLRVDGMDILCVREATRFAAAYCRSGKGPILMELQTYRYHGHSMSDPGVS

GLRVDGMDILCVREATKFAAAYCRSGKGPILMELQTYRYHGHSMSDPGVS

GLKVDGMDVLCVREATKFAANYCRSGKGPILMELQTYRYHGHSMSDPGVS

GLRVNGMDILCVREATKFAADHCRSGKGPIVMELQTYRYHGHSMSDPGIS

301 350

YRTREEIQEVRSKSDPIMLLKDRMVNSNLASVEELKEIDVEVRKEIEDAA

YRTREEIQEVRSKSDPIMLLKDRMVNSNLASVEELKEIDVEVRKEIEDAA

YRTREEIQEVRSKRDPIIILQDRM"gNSKLATVEELKEIGAEVRKEIDDAA

YRSREEVHNVRSKSDPIMLLRERIISNNLSNIEELKEIDADVKKEVEDAA

351

QFATAD?EPPLEELGYHIYSSDPPFEVRGANQWIKFKSVS.

QFATADFEPPLEELGYHIYSSDPPFEVRGANQWIKFKSVS.

QFATTD?EPHLEELGHHIYSSDSSFEVRGANPWIKFKSVS.

QFATTDPEPAVEDIANYLYHQDPPFEVRGAHKWLKYKSHS.

Fig. 3. Amino acid sequence alignment of human somatic (PDHAI) , human testis-specific (PDHA2), mouse somatic (Pdha-I) and mouse testis-specific (Pdha-2) Elo~ sequences. PI, P2 and P3 are phospho- rylation sites. (**) marks the TPP binding site. Mitochondrial import

sequence is in bold type.

polyadenylation signal ( A A U A A A ) and the start of polyadenylation.

The human testis-specific PDH E l a gene is an intronless autosomal gene. To ascertain if an analogous situation exists in the mouse we cloned and sequenced a genomic D N A fragment (mtEla 2.3) containing the mouse testis-specific gene. The genomic sequence was identical to the cDNA sequence from clones 2.4 and 3. We therefore conclude that Pdha-2 is an intronless gene.

Sequence generated in the promoter region (607 nucleotides sequenced from the genomic clone mtEla 2.3) identified an Spl binding site at - 8 1 (Fig. 2).

Fig. 2. Nucleotide and amino acid sequence of mouse testis PDH E l a (Pdha-2) genomic sequence. The promoter region contains an Spl binding site, (GT)(GA)GGCG(GT)(GA)(GA)(CT), which is un- derlined ( ). In the 3' untranslated region two polyadenylation signals, A A T A A A , are underlined ( ). Polyadenylation of m R N A starts at bases marked with an asterisk (*). Sequence recogni- tion element CAYUG is underlined with ( ~ ~ ~ ) . A 'U-rich' sequence is underlined with a broken line ( - - - - ) . Two direct repeats are marked ( # # # ) and ((ct~t;(ctO. Sequence in bold repre- sents a promoter sequence that shows homology with a pgk-2 pro-

moter sequence.

Comparison of human and mouse PDH Ela genes Table II summarises the comparison of the human

and mouse P D H E l a nucleotide and protein se- quences. Coding regions show between 75.5% and 88.6% similarity at the nucleotide level (Table II). However, the nucleotide sequence of noncoding re- gions of the four E l a sequences show no significant homology except for a 28 bp region in the 5' untrans- lated region of the human (PDHA2) and mouse (Pdha- 2) testis-specific genes (Fig. 2). These regions share a high degree of homology (78%) and are located 260 and 259 nucleotides upstream of the translational start in PDHA2 and Pdha-2, respectively.

The primary amino acid sequences of the mouse P D H E l a proteins are similar to those found in the homologous human proteins (Fig. 3). The difference in amino acid composition varies from 2% to 25%. Most of the changes are conservative but it is clear that these changes are not random as more are observed towards the C-terminus. It is also evident that the sequences near the three phosphorylation sites are highly con- served, as is the region near the proposed thiamine pyrophosphate binding site at Trp-214-Lys-215 (Fig. 3).

The precursors to the human liver and testis PDH E l a proteins have typical mitochondrial import se- quences [9,15]. The N-termini of the two mouse PDH El a proteins are also typical mitochondrial import sequences, characterized by the ability to form an amphiphilic a-helix, containing several basic, but no acidic amino acids [16]. Although the import sequence for the mouse testis specific PDH E l a conforms to these constraints, the primary amino acid sequence has diverged from the other E l a human and mouse import sequences. If the mitochondrial import sequence is cleaved at the same site in human and mouse, the Pdha-1 and Pdha-2 genes code for mature proteins of 361 amino acids with molecular weights of 40 135 and 40 085, respectively.

PDHA1, Pdha-1 and Pdha-2 all have two possible initiator methionine residues in the mitochondrial im- port sequence. It is not known which methionine acts as the translational start point.

Sequence analysis of rat and porcine PDH E l a cDNA clones indicates a high degree of homology with the human P D H A 1 sequence [17,18]. Rat and porcine mature protein sequences are calculated to be 99% and 98% homologous to mouse Pdha-1 mature protein sequences, respectively, while the nucleotide sequences are 95% and 88% homologous.

Chromosome assignment In situ hybridization has shown the presence of two

P D H E l a loci in mice, one on the X-chromosome and one on chromosome 19 [10]. In order to assign the

1 2 3 4

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Fig. 4. Chromosome assignment of Pdha-1 and Pdha-2. Male (lanes 1 and 3) and female (lanes 2 and 4) mouse DNA was digested with BglI and blotted. The blots were probed with the Pdha-I specific probe ml PDH 10:1 (lanes 1 and 2) or the Pdha-2 specific probe mt

PDH 1.2a (lanes 3 and 4).

cloned genes to these loci we performed Southern blot analysis. Fig. 4 shows that a band in the lane contain- ing genomic DNA from a female mouse is approxi- mately twice as intense compared to the corresponding band in the lane containing male mouse DNA when probed with the mouse liver-specific probe. This indi- cates that the liver sequence is located on the X-chro- mosome. Furthermore, bands in lanes containing male and female mouse genomic D N A when probed with a mouse testis-specific probe, are of approximately equal intensity. This indicates an autosomal location for the testis-specific sequence.

Discussion

We have shown that two isoforms of the mouse PDH E l a subunit exist. One of these, Pdha-1, is expressed in somatic tissues, whereas the other, Pdha-2, appears to be specifically expressed in spermatogenic cells, as it is not detected in kidney, liver, heart or brain [19,20]. We report here the isolation and se- quence analysis of cDNA and genomic clones coding for thd somatic and testis specific isoforms of mouse PDH E l a .

D N A sequence analysis of cDNA clones has indi- cated that the somatic form of mouse P D H Elc~ is coded for by at least two mRNAs. We have isolated two Pdha-1 cDNA clones that differ in length of 3' untranslated region. One clone has a 3' untranslated region of 143 bp and the other approx. 1.6 kb. The reason for the existence of the two different Pdha-1 mRNAs is not known, but was also noted in the equivalent human PDH E l a gene [15].

Two Pdha-2 transcripts were detected by cDNA sequence analysis. These also vary in size of 3' untrans-

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lated region. One is 108 nucleotides and the other 360 nucleotides in length. The presence of two mRNAs agrees with RNA blot analysis of Pdha-2 expression in spermatogenic cells [19]. However, the expression pat- tern and possible function of the two Pdha-2 mRNAs must await further analysis.

Each of the Pdha-1 and Pdha-2 cDNA clones char- acterized contain a polyadenylation signal that immedi- ately preceeds a poly(A) tail. We assume therefore, that differences in transcript length are due to the utilization of alternative polyadenylation signals.

The consensus recognition sequence e lement A A U A A A (polyadenylation signal) is necessary but not sufficient for polyadenylation. A large proportion of transcripts that undergo polyadenylation contain a ' G U ' or 'U'-r ich element in addition to the polyadenylation signal. Mutation studies have shown that these ele- ments play a role in efficient polyadenylation in many genes when present less than 50 nucleotides down- stream from the A A U A A A (for review refer to Ref. 21). In Pdha-2, a 'U'-r ich element is observed 25-48 nucleotides downstream from the second polyadenyla- tion signal (78% of the nucleotides are uridines). This sequence therefore may be involved in polyadenylation in concert with the second, distal A A U A A A in Pdha-2.

In a survey of 61 vertebrate sequences, Berget [22] observes another sequence element, C A Y U G which often appears between the polyadenylation signal and the start of polyadenylation. In Pdha-2 the sequence CATTG is present downstream from A A U A A A and upstream from the start of polyadenylation (Fig. 2). This consensus recognition sequence is complimentary to regions within the small nuclear RNA U4 suggesting that U4 small nuclear ribonucleoproteins may mediate polyadenylation in a similar manner to the role of U1 snRNPs in splicing [22].

We have isolated a 3.2 kb genomic DNA fragment and confirmed by DNA sequence analysis that it con- tains the testis specific Pdha-2 gene. This revealed that the Pdha-2 gene is an intronless gene, similar to the human PDHA2 and PGK-2 genes. Furthermore, DNA sequencing has enabled us to study the regions flanking the coding sequences in some detail.

It has been suggested that these testis specific genes are functional retroposons, based on the presence of direct repeats flanking the reverse transcriptase- processed m R N A [23]. Two possible direct repeat structures have been detected in the mouse testis- specific gene of PDH El a (Fig. 2). One sequence starts at nucleotide 1351 (5 ' -GTCTTATAAACCTT- TAT-3 ' ) with the similar sequence at nucleotide - 3 3 5 (5 ' -CTCCTACAAACCTCAAT-3 ' ) . The other repeats are at nucleotides 1600 ( 5 ' - G T A G A T T T T T G A T T T - 3 ' ) and - 2 0 8 ( 5 ' - G G A C T T T T T T G A G T T - 3 ' ) . The most 3' repeat in each set immediately follows the possible remnants of a poly(A) tail, the location of which is in

agreement with the cDNA data. We think it unlikely that such repeats are conserved during evolution if they have no function. Furthermore, creation of the intronless processed gene must have happened in a common ancestor. If they have been conserved due to a strong selection pressure one might also expect ho- mology between the repeats found in the human and mouse testis-specific PDH Elc~ genes. However, no obvious similarities were detected. The relevance and possible function of these putative repeats are there- fore not known.

We have compared the promoter regions of the testis-specific mouse and human PDHElc~ genes with that of PGK-2 and with their somatic isoforms. The promoter sequences of the human P D H A I and PGK-1 genes are highly G + C rich, as are those of many other house-keeping genes [24]. However, the G + C con- tents of the testis specific PDHA2, Pdha-2 and the PGK-2 promoters are significantly lower, which might suggest that differences in G + C content affect the expression of these genes [24]. The rooters have regions that are similar sus sequence [25]. In the mouse sequence is found at nucleotide

testis specific pro- to the Spl consen- Pdha-2 gene this

- 8 1 and has the sequence 5 ' - G T G G G C G T G G C - 3 ' . Other similarities between the testis specific promoters include the Pdha- 2 sequence 5 ' - A G T T G T A C A G G G A A A A G A C A C T - CAGTT-3 ' at nucleotide -197 . A similar sequence is found in the human PDHA2 gene at nucleotide 260 ( 5 ' - G T T T A A C A G G G A A A A G G G A C T C A G A T - 3 ' ) and in the human PGK-2 gene at nucleotide - 8 1 1 (5 ' - A G T T G T A C A G G T A G G A A A G C A - 3 ' ). Analysis of the pgk-2 promoter in transgenic mice [26] showed that deletion of the region - 1 4 0 0 to - 5 1 5 reduced expression by approx. 50%. However, the authors con-

TABLE Ill

Percentage corrected diz~ergence of PDH Ele~ and PGK gene sequences

Calculations according to Perler et al. [27]. h-I is human somatic isoform, h-2 is human testis isoform, m-1 is mouse somatic isoform, m-2 is mouse testis isoform. PGK, phosphoglycerate kinase. Replace- ment changes are nucleotide substitutions that alter a codon so that an amino acid is replaced. Silent changes are nucleotide substitutions that change a codon to a synonymous codon and therefore do not change the amino acids.

% Replacement Changes % Silent Changes PDH PGK PDH PGK

h - l / m - I 1.08 1.25 76.4 32.2 h - l / h -2 7.53 6.44 79.2 62.1 m- l / h - 2 6.98 7.54 96.1 72.5 m-2/h-2 15.43 7.75 83.4 65.2 h - l /m-2 14.25 10.25 102.8 96.4 m-2/m-1 14.06 10.54 120.8 103.8

clude that no cis-acting elements which affect expres- sion level and tissue specificity are present in this region.

Specific probes for the testis and liver isoforms of PDH were generated and used to probe Southern blot filters with male and female mouse DNA. These re- sults, combined with the in situ hybridization data [10] show that the Pdha-1 gene is located on the X chromo- some, region F3-F4, and the Pdha-2 gene on chromo- some 19, band B. This is analogous to the situation in humans where PDHA1 is on the X-chromosome and PDHA2 is on an autosome (chr. 4) [7-9].

The molecular evolution of PDH E l a genes can be examined by calculating the relative amount of replace- ment and silent changes. Replacement changes are nucleotide substitutions that result in an amino acid substitution. Silent changes are nucleotide substitu- tions that result in a change to a synonymous codon. The resulting figures are corrected for multiple substi- tutions within a codon [27]. Table III shows that mouse and human somatic nucleotide sequences differ by 1%. However, mouse and human testis sequences differ by 15%. Since the same time period presumably has elapsed for the sequences to change, the somatic iso- forms are more conserved than the testis-specific iso- forms. This indicates that the somatic sequences can tolerate relatively few amino acid changes, presumably because of stringent selective pressures. A similar pat- tern was observed with the human and mouse PGK genes (sequences from Refs. 28 and 29) (Table III). Why testis sequences are much more divergent is not understood.

It is well established that rodents and humans origi- nated from a common ancestor. The relative rate of DNA change for PDH E l a for these two species can be determined by comparing sequence changes be- tween E l a isoforms within each species. A comparison of human liver and testis sequences reveals that 79% of the sequence has experienced silent changes, but in the mouse, a similar comparison shows that 121% (cor- rected) have undergone silent changes (Table III). Silent changes are largely free of selection and reflect the rate of DNA change. Therefore, the rate of DNA change is higher for the mouse PDH E l a genes. This is also true for human and mouse PGK genes. Britten [30] has examined selectively neutral DNA sequence differences to determine the rate of DNA change between taxonomic groups. He observes a faster rate of change in rodent sequences relative to higher primate sequences, and suggests that variation and selection of biochemical mechanisms such as DNA repair and replication probably account for this difference in rate of DNA sequence change.

The expression pattern of PDH E l a in mice ap- pears to be similar to that in humans. The isolation and characterisation of the mouse PDH E l a genes will

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enable us to study the expression and regulation of this subunit in detail, especially in spermatogenic cells and in the fetal brain. The latter is of importance for understanding the clinical effects of PDH E l a defi- ciency and for developing a mouse model for this disorder.

Acknowledgements

We thank D. Kirby and H. Vogel for synthesising the oligonucleotides and Dr. R. Iannello for stimulat- ing discussions and help preparing this manuscript. This project is partly supported by a block grant from the National Health and Medical Research Council of Australia.

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