THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1993 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 268, No. 33, Issue of November 25, pp. 24950-24956,1993 Printed in U. S. A.

Gene Structure of Human Mitochondrial ATP Synthase ?-Subunit TISSUE SPECIFICITY PRODUCED BY ALTERNATIVE RNA SPLICING*

(Received for publication, April 21, 1993, and in revised form, July 15, 1993)

Chie Matsuda, Hitoshi Endo, Shigeo Ohta, and Yasuo KagawaS From the Department of Biochemistry, Jichi Medical School, Minamikawachi-machi, Kawachi-gun, Tochigi 329-04, Japan

We completely sequenced the human gene for ATP synthase y-subunit, which was approximately 23 kil- obases long and was composed of 10 exons. Exons l and 2 encoded the N-terminal presequence required for mitochondrial import, while exons 9 and 10 en- coded the C-terminal portions of mature protein. En- zymatic amplification of human heart and liver cDNAs using the polymerase chain reaction revealed two mRNA transcripts that were predicted to encode two 30-kDa isoforms of the y-subunit, which differed by the addition of a single amino acid at the C terminus of the liver type isoform. These two mRNA transcripts of the heart (H) type and liver (L) type were generated by alternative splicing of an exon. The same alternative splicing event was observed in bovine tis- sue. In human tissues, the H type mRNA devoid of exon 9 was expressed specifically in the heart and skeletal muscle, which require rapid energy supply. The L type mRNA was expressed in the brain, liver, kidney etc. Both transcripts were expressed in the skin, intestine, stomach, and aorta. This tissue specificity of transcript heterogeneity suggests the distinct functional or regu- latory roles of the y-subunit isoforms in the catalysis of ATP synthase. This is the first report on tissue- specific isoforms generated by alternative splicing in an energy transducing mitochondrial protein.

Mitochondria provide most of ATP for eukaryotic cells by oxidative phosphorylation. Mitochondrial ATP synthase (FoF1) catalyzes ATP synthesis, utilizing an electrochemical gradient of protons across the inner membrane during oxi- dative phosphorylation (1-6). F1, the catalytic portion of FoFI, consists of five different subunits (a , @, y, 6, and t ) assembled with a stoichiometry of a#& (5-8). The catalytic sites are located either on the @-subunit or at a-p subunit interfaces. The exact role of the y-subunit in mammals is not known. The y-subunit of Escherichia coli is essential for reconstruc- tion of an ATPase activity and for the in vitro assembly of the F1 portion (6, 8,9).

The mammalian y-subunit, like other subunits of F1, is encoded by the nuclear genome. These cDNAs and their gene

* This work was supported by a Grant-in-Aid from the Ministry of Education, Science and Culture of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession nunbeds) 016561, 016562, and 016563.

$ To whom correspondence and reprints should be addressed Dept. of Biochemistry, Jichi Medical School, 3311-1, Minamikawachi-ma- chi, Kawachi-gun, Tochigi 329-04, Japan. Tel.: 0285-44-2111 (ext. 3149); Fax: 0285-44-1827.

structure have been described in bovine studies (10, 11). In humans, the cDNAs and the genomic genes that encode the a-, p-, and 6-subunits have recently been described by some groups including ours (12-14). For the y-subunit, bovine cDNA and its genomic gene that lacks the 5”upstream region have been reported (11). The human gene structure for the p- subunit and tissue-specific differences in its levels of mRNA expression have been reported (15, 16). Recent analyses of the 5”flanking region of a number of mammalian mitochon- drial genes proposed some candidates of cis-elements that may regulate the coordinated expression, for example, “a respiratory enhancer” (17), NRFl and NRF2 binding sites (18, 19), OXBOX (20), and REBOX (21). To understand the molecular basis of the coordinated expression of the genes for the human F1 complex, it is necessary to analyze the gene structures, tissue specificity, and its mechanism of expression of other subunits including the y-subunit.

Several mitochondrial proteins used for energy production reportedly have isoforms. For example, human adenine nu- cleotide translocators are encoded by three different genes, T1, T2, and T3, which are expressed in a tissue-specific manner (22-25). cDNAs for tissue-specific isoforms have also been detected in the mammalian proteolipid (26) , a compo- nent of Fo complex, and in three mammalian subunits (VIa, VIIa, and VIII) of cytochrome c oxidase (27-29). These iso- forms are encoded by multiple nuclear genes, not generated by alternative splicing. We describe the complete sequence of the gene for the human ATP synthase y-subunit and tissue- specific isoforms of the subunit generated by alternative splic- ing of exon 9.

MATERIALS AND METHODS

Probe Preparation and Genomic Library Screening-Genomic gene encoding the ATP synthase y-subunit was isolated from a human genomic library by a method of plaque hybridization using the cloned PCR’ product of the bovine y-subunit cDNA as the hybridization probe (11). To prepare the hybridization probe, sense and antisense strand oligonucleotides (5’-GCCGCAATGGATCCAAGTTCGA-3’, 5’-CAGCTTGCTGAATTCTTCTTTAC-3’; corresponding to nu- cleotide numbers 105-126, 929-949 in the bovine ATP synthase y- subunit cDNA, respectively) were synthesized and used as primers in the PCR amplification of cDNA from bovine liver. The amplified DNA fragment, length 866 bp, was subcloned into pTZ18R. A human genomic library derived from peripheral blood leukocytes was con- structed into the phage vector, XDASH I1 (Stratagene, La Jolla, CAI, and screened with the 32P-labeled bovine y-subunit cDNA. Phage DNAs were isolated from positive plaques and subcloned into pBluescript or pBluescript I1 (Stratagene). These subclones were used to create unidirectional deletion mutants by exonuclease 111. Selected deletion clones were sequenced by the dideoxy chain termination method (30) using Sequenase (U. S. Biochemical Corp.). The nucleo-

The abbreviations used are: PCR, polymerase chain reaction; bp, base pairs; RT-PCR, reverse transcription-polymerase chain reaction; bp, base pair(s); kbp, kilobase pair(s).

24950

ATP Synthase y-Subunit Gene and Tissue-specific Splicing

E I I I LHATPG23 1

H H

LHATPG21 t H y HE

24951

y kHATPG5

E H

E E H kHATPG2 I

2 kbp . FIG. 1. Schematic representation of the human ATP synthase y-subunit gene. The relative position of exons is shown. Exons and

introns are denoted by black boxes and solid lines, respectively. The newly identified exon 9 is shown by an open box. The relative positions of the phage clones used for mapping the gene are indicated by the lines. E, EcoRI; H, HindIII, restriction sites. Open E denotes polymorphic EcoRI site that presents in clone XHATPG2.

+ I 4-

111 -b IV

4- II

FIG. 2. Amplification of the human ATP synthase y-subunit cDNA. To isolate cDNA clones, PCR was carried out using the primers I (at 5’-arm of pcD2) and 111 (complementary to exon 3 of the human y-subunit gene), and primers I1 (complementary to the 3’-arm of pcD2) and IV (corresponding to exon 8). Sequences of primers are shown under “Materials and Methods.” The DNA extracted from HeLa or HT29 pcD2 cDNA library served as the template for amplification.

TABLE I Sequence of intron/exon boundaries of the human ATP synthase

r-subunit gene

Intron

I I1

111 IV v

VI VI1

VI11 IX

Size

bP 6892 946

1811 576 111

2169 332

4113 639

Sequence

5‘ Boundary 3’ Boundary

ATGgtatggca ATAgtaagtat TAGgtaaggga TAGgtaattta CAGgcaagaca CTGgtaagtag CTTgtaagtac TCTgtgagtaa GAGgtaaagtt

atcctcagGAT t t t t t cagTCA t t t t t aagCTC tcttatagGAC aattctagGTC tttaacagACA gtttgtagCTG ataaccagGGA t t t t t cagGTA

tide sequence of all the exons and introns was determined on both strands.

Restriction Mapping-The phage clones obtained were mapped by single or double digestion with restriction enzymes that recognize six nucleotide sequences. More precise maps were obtained by the restric- tion digestion of fragments subcloned into the vectors.

Hybridization with Human Genomic DNA-Genomic DNA derived from human peripheral blood leukocytes was prepared by the protease K method (31). Each 5 pg of the human genome DNA derived from two healthy Japanese males was completely digested with EcoRI, HindIII, or Sad. Digests were fractionated by electrophoresis on a 0.6% agarose gel and blotted onto a nylon membrane (Hybond N- plus, Amersham Corp.). The DNA fragment to be used as a hybridi- zation probe was produced by PCR amplification of 209 nucleotides, corresponding to exons 2,3, and 4. Hybridization was performed with the probe in rapid hybridization buffer (Amersham) for 2 h. Next, the membrane was washed with a washing buffer containing 1 X standard saline citrate (1 X ssc: 150 mM NaCl, 15 mM sodium citrate, pH 7.0) and 0.1% SDS. Autoradiography was then performed.

Cloning of the Human ATP Synthase y-Subunit cDNA-To deter- mine the noncoding regions of the human ATP synthase y-subunit cDNA, the cDNA fragments, including the noncoding regions, were amplified by PCR using DNAs extracted from HeLa or HT29 (a cell line derived from human colon carcinoma) pcD2 cDNA library as the template. This library was a gift of Dr. H. Nojima (Department of Molecular Genetics, Research Institute for Microbial Diseases, Osaka University, Japan). Primers used in PCR were designed on the 5‘- and 3’-arms of pcD2 vector (5’-GGAAGTGTTACTTCTGCTCT-3’ and 5’-TCTTTCCGCCTCAGAAGGTAC-3’, respectively) and de- signed on exon 3 (5”GGTGTTTCTTCTTGTCTTCAGG-3’) and on exon 8 (5’-GTCATCACAAAAGAGTTGATTG-3‘) of the human ATP synthase y-subunit gene (see Fig. 2). PCR products were sub- cloned into dideoxy T-tailed pUC119 (32), then sequenced.

RT-PCR Anulysis of RNAs from Various Human Tissues-Various human tissues were obtained at autopsy in cases with informed consent. Total RNAs were prepared by the acid-guanidine method (33). The poly(A)’-enriched fractions were then purified via oligo(dT) column chromatography. Reverse transcription was carried out using an oligo(dT),n primer. Amplification of DNA was performed by PCR around exon 9 using a pair of specific primers, sense-strand oligonu- cleotide on exon 8 (described above), and antisense-strand on exon 10 (5’-TAATGGAGGAACAGTTTCTTCG-3’). The size of the PCR products was determined by 3% agarose gel electrophoresis to estab- lish whether the mRNA derived from the various tissues lacked exon 9.

RESULTS

Genomic Structure of the Human ATP Synthase y-Subunit Gene-To isolate the human gene that encoded the ATP synthase y-subunit, a human genomic library constructed in the vector XDASH I1 was screened using the bovine liver cDNA (11) synthesized by PCR as a hybridization probe. A total of 9 x IO5 independent clones were screened, and 12 strongly positive hybridization signals were obtained. Four clones, XHATPG2, 5, 21, and 23, were chosen for detailed analysis since preliminary restriction mapping and Southern

24952 ATP Synthase y-Subunit Gene and Tissue-specific Splicing - 4 2 1 8 S A T G G A C CTCTTACACA ATTTTTGCCT CATCTGTGCC CTATAAGAAT CATAGTTATT ATTTAAATTT ATTTTTAARR GAAACTTGGT ATCAGCCTCA

S a u 3 A I C I E B P R R I -4118 TAAATAAACA GTATCACTTT CCATATATTA AAAAATAAAA TAAAATGAAC AAACAATACA GTTACTAAAT CCTAGAAACC CAGGAGTAGA TTGGGGGGAT - 4 0 1 8 ACCCTGGCTC TGACCTTCAT TCTCTTCAGC CTTCCACTAT CAGGAGATTA CAGAAAAGAA TTAATCCTTT ACTCCTTTCC CAAAAAAATT GCATCTCAGT

Spl ( R I

C / E B P B ( R ) -3918 ATAACATTTA AGGATGGCCA GACACAGTGG CTCATGCCTG TAATCCCAGC ACTTTGGGAG GCCAAGGCAG GAGGATTGTG TGAGCCTCCG AGTTCAAGAC -3818 CAACCTGAGC AACACAATGA GACCTCCAGC TCCACAAGAT ATTTTTTGAA AATTAGCCAG GCATGGGTGG TATGTGCCTA TAGTCCCAGC TACTTGAGG

C / E B P B ( R l C / E B P o

-3718 -GCGG GAGG-T TAAGCCCAGG AGTTGGAGGA TGCTGTGAGC TATGATCGAG CCACTGTACT CCAGTCTGGG CAAGAGTAAG ACCCTGTCTC

-3618 RAARACAAAA AAAAATAAGT TTAAGGATAA CCAAATAGCC CTAGTTGATG AGCAAAAGCT CATTTGTACA AAAGCATGGC AGCCAATCAT AAATGTTAAA -3518 M T T A G AAAAATAACC TCTTTGCAAC TCCTAATAAA ATAACTGACT CAGGCAAGGA TGCTAAAACA CACAGGTAAA AGGTTAATGG GAAACTAGAT

AP2 lR1 C l E B P a S P l

C l E B P a

- 3 4 1 8 ATATAGGTAT GATTTCTAAG TATTACCCCA ATACTCACTT GCTCTTACAA GGGGGAARAT AATACTTTCT AATATTATTT ACARGCTGTC ACCT- - A P 1 , A P l i R l

- 3 3 1 8 F T T T A A G T A CTACCAAACA TTATTGGCTT CCTTATATGA TGCTATATGA GATATACAAG ATCACCTATA AAGAATACTA TAGGCTGGGC ATGGTGGCTT

- 3 2 1 8 GAGCCTGTAA TCCTAGCACT TTGGGAGGCC GAGGCAGGAG GAGTGCTTGA GTTCAGGAGT TTGAGACCAG CCTGGGCAAC ATGGTGAGAC CCTATCTCTA -3118 TACAAATAAA AAAGTTAATT AGGCATGGTG W C A C C T GTGGTCCCAG CTACTTGGGA GGCTGAGGCA G G A G G A G G U T T G A G C T GGGARGGTCA

C l E B P b i R ) APl i R l

SPl

-3018 AGGTTGCAGT GAGCCATGAT CGCGCCACTG TACACGAGCC TGGGCTACCA ATTGAGACCT TGICTCAARR ARARRRARAR AAATCATATT CCTCCCTGCA - 2 9 1 8 AATGTTTAAC CTGAAATTAA TCTATCAAGA TCACCTACAA AGTATTCTTT CTCCCCAARR TGTTTAATCT AACCCAATCA m T T A G ATATAATTTC

- 2 8 1 8 CAGTTGACAG AAACTACGAG TTAGAAGGCA AAAGACAACC AGAGCAAACA AGTCAACATA TATGGGATGC TACAGGACAA CAGCCTGGCC TCCTCTCTTC - 2 7 1 8 AACAAGTCM TGTCAAGAAA AAGAAGGCTG GGCGCAGTGG TTCACACCTG TAATCCCAGC ACCTTGGGAG GCCGAGGCAG GTGAATTACC TGAGGTCGGG - 2 6 1 8 AGTTTGAGAC CAGCCTGGCC AACGTGGCAA AACTTCGCCT CTACCAAAAA TACAAACATT AGCCAGGTGT GGTGCCAGGC ACTGTAATCC CAGCCTGTAA

C I E B P Q ( R ) SPl C l E B P a

HindIII

S p l ( R I - 2 5 1 8 AGTAGCTGGG ACAGCTACTT GGGAGGCTGA GGCATGAGAA TCACTIGAAC CCGGGAGCAA AGGTTGCAGT GAGCTGAGAT CGCATCACTG CACTCCAGCC

- 2 4 1 8 TGTGAGACAG GTGAGACTCT GTCTATAAAA AAAAAMAAA GAAAAACGGG TGGAGGGAAA ATGGGTTATA TTCTAAATTC AGAATATAGA GRPAATTAAA - 2 3 1 8 TTTGGAGAAA ACTAARAGTC CTAATAACTC AGAGCACAGA GGACTTTTTG GGCAGTTAAA CTATTTTGTG AGATTTTATA ATGGTGAATA CGTATCATTA - 2 2 1 8 TGCATTTGTC AAAACCCATA GGATGTACAA CACCAAGAGT GAACCCTAAT GTAAACTATG GATTCTGGAT GATACTGATG TGTCAATGTA GTTTCATCCA - 2 1 1 8 TGGTAACAAA TATAACACCT CGTTGCAGGA TGTTGATGAT GAGGGAGACT GGAGAGGGGA GCGGAGGTCC TAATAACCAA ACCTATAACA G T C T G T

SP 1

- 2 0 1 8 TTTGATCGAA TCTTGATTTC AAGGAGCAGT TGGAGGACAA CTGAGGAAGT GTGAATGTGT ATTAGATGAT AAAGAGGAAC TTCTATTAAT TGTGTTATGT -1918 TGATGACATA TACTATGGTT ATATAGAAAA GTATCCTGAA ATTTTAGAGA TGCCTATTTA AGTATTTAAA GGTGAAATAT CCTGATATTG GTATTTACCT -1818 TWTATTT TTTAAAAAGT AMTACGGCA AALRTTACTTA AAGTGTTAAG TCTGGGTGTT ACAGACTGCA TGTCTGCGTC CCCTCAAAGT TCATATATTG

- 1 7 1 8 ~ C C C T A A C C CCCAAGGGAA TGGAATTAAA AGGTGGGCCT TTGGAGATGA CTGGATTTAG ATGAGGTCAC AAGGGTAGAG CCTGCATGAT GAACAGGGTG

-1618 GGACCGTATC TCAAAAAAAR AAAGAGGAAG AGAAGTTTCT ATTAGAGCTT GCTCTATCTG CTTTGTGAGG ATAGAGGCAG TTTCGCAAAC CAAGRRGAGG -1518 ACCCTCACTA GAATCTGACC ATGCCAGCAC CTTGATCTTG AACTTCCCAG CCTCCAGAAC TGTGAGAAAT AAATTTCTGT CATTTAAACC ACCCAGTCTA

- 1 4 1 8 CTGCACTTTA TATGGCAGTC CAAGCAGACT RAGACATTAG ATAATGCATA TACAGGTATT CCTTAATAGA ACTAAAGAGA GTRRCTGTAT CAATATTAGT

C l E B P a

C / E B P B ( R I

AP2

S p l ( R I

C / E B P b ( R l

-1318 ATGAGTCCTA CCTMAATAC CTCTTCCTTC ACTTGCAGAA ACACTGGATT AGATTATTCC AATTGCCTCA GTGTTCTTCC ACCCTTACCCCCAGTZ

-1218 SCTCCGGA GAAAAATATG ATCACATCAC TAGCCTGCAC AGCATTCAAT CAACCACCTC TTTAATGGGG AAAAGTGTAR ATTCCACAGT GTAGCCATAT

- " - C I E B P ~ W I C / E B P P ( R I C = P P ( R I C l E B P e (Rl

-1118 AAGGCTTTTC GAGATCTGCC CAGACTCCTT GAGCCACTCT ATAIATACTT CACTCATTTA AAACTCAGCG ACAATCCATC AAGTAGGTGA AATTATTATT -1018 CCCTCTTTAC AGATAAGTAA AGGGGAATAT AAGTAAAPAA CTTGCCCAAG ACCACACGGT TGGTAAGTGG CCCTGCTTGA CTTTAAATTT TGACAGTCTG

-918 ATATATCCAT AAAcCAGATA AATCCATAAT TTTAACCACA ATATTATTCT GTGCCCGGTA CAGTGTAGGT GCTCAATAAA TGCTTGCTAG ATGAGTAGTG

- 8 1 8 TGGARGAGCT TAGcTCTCCT TTTTCACCTG ATCCCAAAAG AGGAGTGAAT CCTAACCACC CTGTTTCTCT TAAGAGTCRR GTCAAGTCAG AGCTGATGAC

-718 TGTTACTGGC =cCGGG CGTCTAGT-AGTATCC CCGCCCTGTG AGGGGTGCAA AACCTTTTCT GATTTCATTT CTTTGGTGGT GGGGATCGGA

-618 CTGGGGAGGA ATGAGGGGTC GCAGAACACA GGCTGCCCGC TGGGCCCGCG GGCTGCGGGG AGGGACGGCT CTCGTGCGCT GAGTAGTTGA CTCGCCCACT

C / E B P p ( R I

Mr3 (Rl

C l E B P I R I

C I E B P a C l E B P a AP 2

SPI ( n )

FIG. 3. Nucleotide sequence of the human ATP synthase y-subunit gene. Nucleotide residue +I denotes the first methionine codon (AUG), the nucleotides that preceded it are negatively numbered. The restriction sites used in the four overlapping DNA fragments are shown. The location of exons and the deduced amino acid sequence are enclosed within boxes. The first nucleotide of the boxed exon 1 corresponds to the 5' end of pcDZ-HeLa cDNA. The Mt3, NRF1-like sequence and the putative Spl-, AP1-, AP2-, AP3-, CCAAT/enhancer binding protein, and F-ACT1-binding sites are underlined or ouerlined. The complementary sequence of these elements is shown in ( R ) . "CCAAT boxes are underlined twice. The direct repeat, which lacks the XHATPGZ genomic clone, is shown as a bold line. Alu family DNA repeats are underlined, and two polyadenylation signals are underlined twice.

blot analysis showed they contained the entire gene for the was tentatively identified following mapping with two restric- ATP synthase y-subunit together with substantial 5'- and 3'- tion enzymes, EcoRI and HindIII, and 32P-labeled fragments flanking regions (Fig. 1). of the 5'- and 3'-portions of the amplified bovine cDNA. Each

The location of exon sequences within the genomic clones fragment that had been digested with EcoRI and Hind111 was

ATP Synthase ?-Subunit Gene and Tissue-specific Splicing 8 8 3 GCATATTAGA AGGCCCGGGA ATGGGCAGGT AAAAGTTGCT T'I'GTGGG'TCG TGCTGTTCAG T T T T G m G A C G G A A T C TGGAAGAATT CAATCCCTGC

9 8 3 CGTCTGAATG CAGCCCTTCA CTGGCTACAG AGTCTCTCI'C TI'CPJACAGT ACTACACTCT GCTGCCTCAC ATTTTTTACA TTTATTAGTA AATATCTGCC 1 0 8 3 CTCATGGTCT CAGATGACAA TTTGTCTTTT CTCTCCTGAA ATGCCATCTC CTTATCGGTA TCTGTAGTTT ATGACTACTG ACACGTTATT U C A G T 1 1 8 3 TCCATTTTAG AGACTACTGA ACTCTAAGAA ATAGAAATGT TTTCCAAATT TGAAGACAGT AAAATCCGGG ATCTTCTCTC AAATTTTTAA TTTCCATTTT 1 2 8 3 CTTATATGCT GCTTTAAAAA AAGGTTTAAT TTTCAAAACG TTAGAGTATT TTGCCGATAC CCTAACAACT ATTAAAATAA CCACTGTGAA TTTAAGTAAG 1 3 8 3 CCTAATGTTT TTATGTTAGC ATGGTAGCCT CTCTGAAAAC AAAAACTAAA ATAACAAATT GTACTTTAAA AAACTTCCAG TAATCAAGAC AGAAGCATGG 1 4 8 3 AAATATGCCT CTTCATGGTT CGTTAAGACA AAGGTCTTTT CTTCCCCTTC CAAATTAATT CTGTGGAGTG GTTTTAATTT C T U T G ATTGCCACTT

1 5 8 3 AGCAGCTCTG TGACCTTGGG CAGGTTACTC AGTGTCTGTC CCACCAGTTC CTCATCTGTC AAATATATGC AATAAAAGTG CACAATTCAG TGGGTTAGTG 1 6 8 3 TGAGGATTAG AGTATTTGCA AAGCATGTAG GACAATGGGA GAAATGCTGA TAGGGATCCA AATGCTGTAT ATCAGTAGAG AAATGAAGAG TTTTTAAGTA

Hind111 C / E B P n o R i

EcoRI

24953

1 7 8 3 CAGTTTCTAC ATCAAATGTA ATGTAAGAAG ATTGCTTGGA CTTGCGAAAA GAGGAAAAGA AGATGCAGTC GGGCTAGGCG CAGTGGCTCA CGTCTGTAAT 1 8 8 3 CCCAGCATTT TGAGAGGCCG AGGCGGGCGG ATCACAAGGT CAGGAGATCA AGACCATACT GGCCAACATG GTGAAACCCT GTCTGTACTA AAAATACAAA

2 0 8 3 TGAGATCGTG CCACTGCATT TCAGCCTGGC GACACAGTGA GACTCCATCT CAAAAACAAA AAAAAAGATG ATGCAGTCAT GGTGTGGGTT AGGTAAGTGT 1 9 8 3 AATTACCCAG ACGTGGTGGT ACGCACCTGT AGTCCCAGCT ACTCGGGAGG CTGAGGCAGG AGAATTGCTT GAACCCCGGG AGGCGGAGGT TGCAGTGAGC

2 1 8 3 GAAGGAGTCA GTTTTTGCTG GTGGCAGGTA ATGGACTTTT ACACGCTTAA TATCAGAATT AGAGTCACTT CATAATTTTC GTTGAGAATT AGCCAATATT 2 2 8 3 GCTTGCTTTT TAAGTTTTTA TGTATCTTAA TATACTCCTT AAAAATCTCT CAAACAATAG TTTCTTGGAA GGGTGTCATT CCTGACTCCC TCCTCCACTG 2 3 8 3 CCTTCGTCAG CTGTTATAAC AGCGCTGTGG AATCATGGTA GATAGTGGTA ATCCACGGTC CATTTGATTC TCCCTCCACT GTGTTTCTTA GTCTCCCGAG

2 5 8 3 TCACTGTTCT TACTCAAATT GTACCTAACA ATTAAAGTAG TAACATAGAC GTCTTCAGAA TTAAATGAAA GCTAAACATG AGAGTGCTTT AAAACACATG 2 4 8 3 GATCTCAGGA CGTGCAATGA TTCAGAGCTT TACCTCCTCA TTTCCAAAAC TTTTATATTT AATAAAGGAA GCTATCCCTA AAGAGAGTTC AATTRGCTCA

2 7 8 3 ATATGGTATT CAATTCAAAT TTATACCATA TGGTATTCTC TGACTAGAAA CTTGTGTCAG TTGAAGTGTC TGCCTAAAAC AATARTATCC AAGAGTGACT 2 6 8 3 AAATTAGGGC AAATGCGTAG CATTATCACA AAAGATGCTA CCTCTTCATA CCTCTGACTT GGAGTACTTA AGGAAACCAA AACATTCTGT GTTTGTCCAA

2 8 8 3 TGTTATAACC AATAAATTTT GTGTTCAGGG AGAAAAGTCA TTACTACACC AAAAAAGGAA GGTTTGACCA AAAACTGGTA TTACTTTTCA AGTGTGATTA 2 9 8 3 TGAAAAGCTG TTGTAGTAGA ACAGTCATGG CAGGTAGTTT GTGCTCTGAA ATAACAGAGC TTGTCAGGAT TTGCCAGCTT TTATCTTTTC CTITAGTAGA 3 0 8 3 TTCATTCTTA AAACCAAACC TAATATTTTT CGTGCTGTAG TGGGAGAATC ACCTGAGCCC AGGAAGTTGA GACTGCAGGA AGCCATGATC ACACCACTGC 3 1 8 3 ACTATAGCCT GGGTGAGGGG AGTGAGACCC TGTCTTCAAA CCGCAAAAAA CAAAGATCCA CTCTTTTTGT TATACAGTTC CACGGATTTG GACAAATGCA 3 2 8 3 TAATGACATG TATCCACCAT TACAATATCA TGCCAAAGAG CTTCACTGCC CTAAAAATCC CTCCTCTACT CTCCCCAAAA CCCTGACAAC CACTTCTCTC 3 3 8 3 AACTATTGCT AGATCCTCGG CTTTTCTGGC ATGTCACATA GCCAGAATCA TACAGTATTT GCTTTTTTTC AGATGGCCTT CTCTCCCTTA GCAGTATGCA 3 4 8 3 CTTAAGGTTT CTCCATGCCT TTTTATAACT TAATAGCTCC TTTGTGGTTT TGGTGAATAA TAGTCCATTG TATGGATGTA TCAGAGTTAG ATTATCCATT 3 5 8 3 CATGTATTGA AGGACATCTG GGTTGCTCCC AACTPT?'GGC ATCATCAAlG AAGCTGCTTT ACATGCTCTT GTGTAGGTTT TATGTGGACA TAACATTTCA 3 6 8 3 TCTCAGTTGG GTAAGTTAAG ACTAGGTTTA GGTTTGTAAA AAGC'I'ACCAG AACATCTTCC CAAGTGGCTG TGTACTATTT TACATTCTGC CAGCCATCAA 3 7 8 3 TGAGAGTTCC TTTTGCTCCA CATCCTCATC ATCATTTGCT GGTCAGTTTC TTGGGTTTTA TCCATTCTTG CAGGTGTGTA GTGATATCTT ACPGTTTTAA

4 4 8 3 GTCATGGATC ATGCTTTTGG TGTTGTGTTT AAAAACTCAT TGCCAAACCC AAGGCACCTG GATTGTCTCC TGTGGTATCT TGTAGAAGTT TTATGGTTTG 4 5 8 3 GCATTTTATA TTTAGGCCTG TGATTCATTT TAAGTTAATT TTTATTAAAT TTGTAAAGTC ATTGTACAGA TTAATATTTT TGCATATTAG TGTCCAGTTG

4 7 8 3 GTGTCAGTCC TGACAATATT GTTTGACTTT TCTAGGTCTT TGGCCTTCCT ATATAACCTT GAAAATCAGT TTATTGATAT CCACAAAATA GATATCAACT 4 6 8 3 TTCCAGTGCC ATTTGTTTGT TGAAAAGAAT ATCCTTTTTT CCTTGAATTG CTTTTGCTTT TTTATTACTG TAACTTTAGA GGAGGTCTTA ACGTCAGGTA

4 8 8 3 AAAATCAAAC TTGCTGAGAT TCTAATGGAT TTTTAAAATT GTAAAACACA TGGAACATGA AGTTTATCAT TTTAACCATA CAATTGTGTG CGGGCATTTT 4 9 8 3 TAACTTCATA CTCCAGTAGT TCAGGGCTGC TGCAGAGGAA TGCAACTAGC TTTTTATATA CACTAACCTT ATATCCTGCA ACCTTGTCAT AAATGCTTAT 5 0 8 3 TGGTTCTAGG AGGGGCTATT TTTATTCTTT GGGATTTTCT ACATAGATAA TCATGTCATC TATGAACAAA AACAGTTTTA TTCCTTCCCT TCAAATTTCT

5 2 8 3 ATGGGGAAAG CTCCTAGCCT CTGGGATTAA ATCAGAGATT TTCTGTCTTG TTTTTTTTTT TTAGCAGGTT GAGGAAGTTC TCTATTCCTA GTTTGCTGAG 5 1 8 3 ATGCCTCTTA TTTTCTTGTC TTACTGCATT AGCTAAGACT TCCAGTATGA TACTGAAGAA GAGTGATGAG TGGGGATTTC CTTATCTTGT TCGCAATCTT

5 3 8 3 AGACTTTATC ATGACTGGGT GTTAGATTTT GTTGGATGCT TTTTCTGCAC CTATTGATTG TATGATTTTT TCTTTAACCT GTTGACGTAA TAGATTATAT 5 4 8 3 TATTTGATTT TTTAATATTG AACCAGCTTT GCATACCTGG AGTAAATCCC ACGTAGTGAT GGTGTATAAT TATTTTTAAT ACATTGTTGG ATTTGATTTG 5 5 8 3 CTAATACACT GTTGAGGATT TTTGCCTCTA ACCTATGCTC ATGACACAIA TTAGTCACTG TCCACCAAGG TCATTCAATT AGAGAAACCT TAGATCATTC 5 6 8 3 TTACTGCTTT CCTTTCTACT ACGAATGTAC ATGTGAAGAA GTTAAATGCT TGCAAATCTA GTCTCCTATT GCTTTGCCAG CATCTTCATC CAGGTTCTTG 5 7 8 3 AGGTCTCTCC TGAGGCTGTG GCAGCCTACA TCCCCATGTC TTCTGTGTTC ATCAAAACTC CAACCTCATT TTACGCTTGT CACTGCCATG TGAGATACTT 5 8 8 3 TGCTGAAATA CTAAACTGAT TTGTGGGCTT ATAAACATTC ATTTCCCTAT TAACCTACAA ATAAGATTTA AACCAATTTG CTTTTTTTGA TATTTAAAAA 5 9 8 3 TATTAATTAT TTAAAGTAAC AAACCCAACT CATGTCTCCA GTTTCCCCTT CTCCATTTCT CACTCTTTCC TGAACATGAA TGCTCTTAAA ATGACATTCT 6 0 8 3 GTTCCCGAGA ATCCCCTCCT CACATCTCTA CTTTGTGCAC ACATTTCATC ATGCTCAGTT CTCCTGTGTA GCTTTGCCTG CAAACACAGG CACTTTGTAT 6 1 8 3 GGATTTGAAG GCAGAAAGAT CATCTAATAT TTTTTAGCAT GTCTTCCCCC ATTAGGCCAG S T C T C ATATTCATCT TTCACCAACA TTTCATTTAA

6 2 8 3 GCCTAATCAT TTTGACTTCT GCACATTAGC ATTTCAATTT TAAAGACTTG AAAAAAAATA CTAAATGTTA TGTAAATGTA ATTTTTTATG AACCTTGTTT 6 3 8 3 ACAAGGTAAC AATATGTCAG AGACCATGTC GTGTTTACCA GTGCCTGGAA TTTAGAGCAT CTTGGTAAAT GTTTATTGAG CTAAATTGPA AAAGTTAAGA 6 4 8 3 ATCAGTTTAT ATAAACTACT TTTACACGTG TAGTCATGTT CGACTATATC ATTGACTCTG TTACTGGAAG AGGTTGCTGT GAACTAAGGA AATTTTTAGA 6 5 8 3 GTGGCTGGCC TTGATTCGAA GTATAGTTGG GCAGCTTAAC AACGGGAGCG GCTTTTCTGT CATGGAAACA AGATAAGCAA AGATATGGAG GTGGGACTAG

6 7 8 3 TAAGGGATTT AAATATATGT CTATTCATRT ACATTCTGAG TCCTGTATTF GATAGATTGT TTCTTTATGG GGTATTCTTT AAATAAATAT ATATTAACTG 6 6 8 3 AGCATGTGTG AGTCAATAGA GCATGTTGAC TTCTGGGAAC CTGGTGGTAC AGACAGGCCA AATAAAAGGA GGCCTTCAAT ACAGGGATTC GCGCCACTAC

EcoRI

6 8 8 3 AAAGTTTTTC TTGTATAATG GAACTATTTT TCAAAAAACT GAAACATTTT TAAAACTTTT ATCCTCA A TTCAAGTTCG AAATATGGCA ACTTTGAAAG

6 9 8 3 AT TAAGTA TGTTGTTTGT CTCAATATGT GAATTCAGAA AAACTAAATT TTGACAAATA CAAATACTTT CAAAAATATA CTGCTTTAGC CCGTTGTGTA

7 0 8 3 TTTGATCAAA ACTGACCAAA TGGATTTCCA TTGTATGTTT GGGACATTGG ACTTGTTCTG AAAAGACCAG TAAGAAAGGA CACCTGAGAC AGCTATGTTC 7 1 8 3 TGTTGTTXGT AGAATGATCA TGATATAACT AGAAAARATT ATACCAGTGT TGGTGTAGGT GGGTGTATGT GTGGGAAGGG GTAARTCATG TTATATATAA

Exon2

I3 i P V R N M A T L K

7 2 8 3 ATAATGTTAT GGAATATGTG GTTAAGGCAA CTTTCAATAC CATTTAATGT TTTAAATTGT ATATTATTGT CTTATGTTGC CATTTGCTTG TCCTAAGIAG 7 3 8 3 TAGCTAGTTT TGCCTTCCTT TTATTAGTAC TATTTCTTTT TTTTTTTTTT TGGAGATGGA GCCTCATTCT ATCACCCAGG CTGGAGTGCA GTGGCGCCAT 7 4 8 3 CTCGGCTCAC TGCAAGCTCC GCCTCCCGGG TTCACGCCGT TCTCCTGCCT CAGCCGTTCT CCTGCCTCAG CCTCCCGAGT AGCTGGGACT ACAGGCGCCC

7 5 8 3 GCCACCACGC TCGGCTAATT TTTTGTATTT TTAGTGGAGA CGGGGTTTCA CCGTGTTAGC CAAGATGGTC TTGATCTCCT GACCPCATGA TCCGCCCACC 7 6 8 3 TCAGCCTCCC AAAGTGCTGG GATTACAGGC GTGAGCCACC GCGCCCGGCT ATTAGTACTA GTTCTAATAT TAAGACTGGT TTAGCATCGT GCTGTTTATT 7 7 8 3 GTTTATCTAA GCGCTCATTA TTCTGCCTTG CACTTTAGAA AAATKICTCA ATAAATTCAC AAGGAAGTTA TACTGTAATT CCTTTGTCTT AACACAGTAC

AG GAGACTAAAG TCCATCAAAA ACATCCAGAA AATTACCAAG TCTATGAAAA TGGTAGCGGC AGCAAAATAT GCCCGAGCTG

CT CGAATATATG GATIGGGATC TTTA TAAG GGAAGAGTGT AATTCACAAA TTAGGAAGAA CTGTTTCACA CAAGGAAGAC

8 0 8 3 TACTGATGAC TTACATTTAC AGCTTACAGT GATGTCAAGC CTATCTGAGC ACTTGCCATG TACTTATTTT TCATCTACAT CCACTTGACA CGTAATGATA 8 1 8 3 ACAACCCGTG TAGGTAGTAC AGGTAGGGAC TGTTAGAATC CCTGCTTTCC AGTGAGAAAA ACTGAAGCAC ACAGAAGTTA AGTAACTTGC CTF,&GATCAC

8 3 8 3 ATTCTGAAAT GTTCCCAGTT GTTAAGTATA CCATCAGAAG AACTTGGAAA CCGTGCGTTA GATGTAAGGG CTTAATTTCA TCCTATGACC TTGTAGTGTG 8 2 8 3 ATATAAAAGG ATGAAGAGCC TCTGTTTGGA CCTATGTGGT TTGG'TGTCGA GTCTGTGTAT TTGTTAGCCA TTATACCGTG ACCATGTTAA C T T T T W T

8 4 8 3 GCATTTTAGG ACTGCAGCGG GGGCGGGTGA GGGGCAGTGC AGTGGAGAGC TGGCCTTGTG GTCATGGATA TGACTCATGA TGGTGTTTTG AGAATCCGAA 8 5 8 3 CGATGTTGAA GCAGGAAAAG CAGCTGAGAA CCTCCATTTT CAAGATAGTT AATCACCATT CTTATCTTTT CAAAAGCATG AAGACATTTT TCCTTCAGAA 8 6 8 3 ATTTATGTAA TCCATCCTTA ATTAGTCTTC TTAATGGAAA AGAGATGAGA TATGACAGTT TAAAAAGAAT TTATATTGTG TTTTTAGATG CTTTATWTT 8 7 8 3 GTTTGATTAT GAATTTATCT TGGAAATAAA GTTCTAAAGA GCTGGAAATG GGAAAGAAGT TTCTATTTTA ACTTGTAATT ACATTCTTTC T C T T A A ~ T T

FIG. 3"continued

T R R L K S I K N I P K I T K S M K M V A A A K Y A R A

E R E L K P A R I Y G L G S 1.

subcloned and then sequenced. Complete nucleotide data obtained from XHATPG23 showed that the clone carried from exon 2 to exon 8 based on homology with the bovine genome and cDNA sequence. To identify the entire exon sequence, the human ATP synthase y-subunit cDNA was amplified from a HeLa or HT29 cDNA library constructed in pcD2 vector. These amplified fragments were then subcloned and sequenced (Fig. 2). The entire sequence of the human ATP synthase y-subunit cDNA revealed that its nucleotide se-

quence closely resembled that of bovine heart cDNA, with the exception of the inclusion of a novel exon (exon 9) that was 37 nucleotides long. From the results of hybridization analyses with the amplified human cDNA fragments, the genomic clones of XHATPG5 and 21 were shown to contain the 5' end and 3' end of the gene, respectively (Fig. 1). The inserts of the phage clones were subcloned and sequenced. The complete sequence of the exons was determined in humans for compar- ison with the human ATP synthase y-subunit cDNA se-

24954 ATP Synthase y-Subunit Gene and Tissue-specific Splicing 8883

9083 8983

9183

9383 9483 9583

9283

9683 4

9 7 8 3 TTATTGTATT TAGTGTATTG CATATAAAAA MTTACTGCT TTTG'ITTGTT TTTAA GTATGAAAAA GCTGATATCA AGGGGCCTGA AGACAAGAAG

98831AAACACCTCC TTATTGGTGT GTCCTCAGAT CGAGGACTGT GTGGTGCIAT TCATTCCTCC ATTGCTAAAC AGATGAAAAG CGAGGTTGCT ACACTAACXC L Y E K A D 1 K G P E O K K

K H L L I G V S S D R C L C G A I H S S I A K Q M K S E V A T L T . ._

9983 ICAGCTGGGAA AGAAGTTATG CTTGTTGGAA 'TTGCTGACAA AATCAGAGGC ATACTTTATA (FITAATTTAA ATATATATTG TTTATTTTCA TAAAGATGTA'

10083 AGCACGAATA AATCTTCTCT CAAAAGTTTT GACAAACTCT CAAAACCTTC ATCAATAACA AGGTATATTC TTCAGGGCCT TTTTTGGTAT ATTCACAAGG 10183 GGGTGTTTGT TTCCTTTTTC TTTTTTCCAG GATATATIGC AACATTTCTG TTTTCCTTCC CCTGTTTTCT ACCTTTGCTT CTTCATCCGG ATCATAAGTT 10283 GAATGCCATG TGAATATCAA AAGCTATCAG CATTAAGCCA AAIGAGGCAT CTGAGGAGCT GTTACAGCCT GGAAATGTTA GCAATAGTTC ATTAGGATTA 10383 GAAGGAGAGG AATAAAGGGG AAAAGAATAT AAAACAGACC TCATTTTTAG TTCTTAGTTC TTTGCAGGTG CTGAAGGAAA GGAATTATGA TAGTGTGTGG 10483 AACAACTCAG GAAATCACCA CCACCCATAC CCCAAAAGAC CTGATCCATG CTGACAATGT TTTCTCCTGC CTGTCCCCTG TGCTCTTTCA GCAGAAATCT

I A A G K E V M L V G I G D K I R G I L Y

10583 TATTAAACTA GCTATGTGAG CTCTTGCTTT TCTTATA A CTCATTCTGA CCAGTTTCTG GTGGCATTCA AAGAAGTGGG AAGAAAGCCC CCCACTTTTG Exon5

10683 GAGATGCGTC AGTCATTGCC CTTCAATTAC TAAATTCTGG ATATGAATTT GATGAAGGCT CCATCATCTT TAATAAATTC ' ~ H S U Q F L V A F K E V G R K P P T F

10783 T C A A A A T TTTATGTTCA TGCTTTTGTT CATATTTIGA AATAACAGTT TAAAAATTAT TTTGAGATTT ACATTTGTTT G D A S V I A L E L L N S G Y E F D E G S I I F N K F

Hind1 I I 10883 ~ T C T C C T A T A AGACAGAAGA AAACCCCATC TTTTCCCTTA ATACCCTTGC AAGTGCT$T AAGTAGTTTT TCTATGATAC ATATTTTTTG GCATATAGAA

1 0 9 8 3 TGGAGAGATT TGTACACTAA GGCAGACACT GTCAAGATAT CTGGTGCTAG CTATAGCAAA TGATTTGGGG CTGTTTCTTT TTTAGATGGA ATCTTGCTGC 11083 CTCCCAGGCT GGAGTGCAGT GGCACCATCT CGCCTCACTG CAACYCCGC CTCCTGGGTT CACACCATTC TCCTGCCTCA GCCTCCCTAG TACCTGGGAC 11183 TACAGCACCC GCCACCAAGC CTGGCTAATT TTI'IGTATT? TTACTAGAGA CGGGGTTTCA CCATGTTAGC CAGGATGGTC TCAATCTCCT GTCCTCGTGA 11283 TCCACCCGCC TCGGCCTCCC AAAGTGCTGG GATTACAGGC ATGAGCCCAG CCTTTTATTT TTTTTTTTTT TTTTTTTTGA GACGGAGTCT CACTGTGTCA 11383 CCCAGGCTGA AGTGCAATGG CATGATCTCG GCTCACTGCA ACCTTCGCCT CCCGGGTTCA TGCCGTTCTC CTGCCTCAGC CTCCCRRGTA GCTGGGATTA 1 1 4 8 3 CAGGTGCACG CCACCACCCC TGGCTAATTT TTG'IATTPTT AGTAGAGACA GCGTTTCACC GTGTTCGCCA GGCTGGTCTT GAACTCCTGC CCTCAAGTGA

11683 CATGATACTT AATTCAGTCA TTGTTTGATA ACTCCTGCTC TArTCTGTCA TTAATTGARA AGTCAGTATC TATTGAGGTT CATCATTAGA TTTTTTTGTC 11583 TCCACCTGCC TTGGCCTTCC AAAGTGCTGG GATTACAGGC AIGAGCCACT GCACCCGGCC GATCTGGGGC TGTTTCTGAG AAGTGCCATC TCTTGACTTC

11783 CTCAAGGATT GAAGAGCGTA GCTAGAAACT GGCTTTGCAA TTTATCIT'TG GGACCTCTAA CTACAATAAA ACRGAAGCTC TAGAATAGAG AGTTGAACAG 11883 TGTTCACAGC TAATTTAcAT TGACTCMTT CTCTTGTTI'T ATTGTlGGAA TAAAGTCATC ATTGATCATT GAAGCATCCT GGAATAATAT TCTGGAACTG 11983 ATTTTAGTTT TCTAAGGCTT TTTTCCCTTG TATAIACCAT CTATTTTCTA ATACTTCATT TATTTTCAAG TATGTACTTT ACACGTTAAT GTTTCTGARli 12083 TTGAGATGGC TCTACCAGCC AATGGAACAT CA1"ITATAAT TGAGTACATI TTTTTCTCTC TTAATGAGAA ATAAAGTAGG TGTTACAACC AGTACCATTT 12183 AATGAAATAT AGTATTTTGA TAATATATAG TACTGTGTCA CCACTTGACC CCAATGAATG AATTTTGAAA TGTACTTCTA AATTTCTACA ACATTI\ARRC 12283 TACGATTCAT ACCAATTTCT AGTTCTCCCA CTCTTTCAIG TGCTTTCAAA GGTTTTCCCT CTCTCATTTT CCAGATTGAT ACATTCAGAT TTCTTCAGTA 12383 ATCAAATGAC TAAGTTGGM TTACTGCACA GGTTGAGTAT CCCTTG'IGTG AATGCTTAGG ACCAGAAATG TTTGGGATTT CAGAATATTT TCATATTTAT 12483 AATGCAGTAT CTTGGGGACA GGRCCTGAGT CTC'TACACCC TAIACACATA GCCTGAAGGC AATTTTATGC AATATTTTAA AGAATTTTCT GCATGAARCA 12583 AAGTTTCAAA TGCACCCCAT CACACAAGGT CAGGTGTGGA AITTTCCACT TGTGCATCAC GTCAGTCTCA AAATGTTTCA GATTTTGGAG CACTTCAGAT 12683 TTTGGACTTT GGGATTAGGG ATGCTCAACC TGTATTACAC TAAATTCGTT TCAATTCATT ATTATCTTTT TTACTGGAAA AAGCATTGAG TGCTTGCTGG 12783 CTTTGTAACA GGCACATGCT AATGCTGTCC ACAGGCCTlC ATAACCACGA GATATAGTGC AGTTATTTCA AATGTTGACC TAATTTTTCT ARRGTTACAC 1 2 8 8 3 ATGTGGAGTA GAATATCAAA TGGTTTATTT ACACATTATA T'WTGCTAAA AATATGCTAT CATCTGTAGT GTGGAAATAG ATGACATTAT TAGAACAGAT 12983 TCATTTACCT TATCTAGCAT TAATTTCTGC TCTGTMTTA 'TAAAGGAGTT TTACACTGAA AAGCTGTTTT TGATGAATTA CTATTCCTTC TATATAACTT

I S Y K T E E K P I F S L N T V A S A 1

13083 GAAAGAAACT CATCACTTGT TTTAACA C AGCAI'GAGTA TCTA?CACGA TATTGATGCT GACGTGCTGC AAAATTACCA AGAATACAAT CTGGCCAACA

13183 TCATCTACTA CTCTCTGAAG GRGTCCACCA CTAGTGAGCA GAGTGCCAGG ATGACAGCCA TGGACAATGC CAGCAAGAAT GCT TAAGT ACACAGTATG

13283 GACAGTGCCA GCAGGAGTGC TTGTGAGCAC ACAGTGAATC TCAGCTGAGA GGAGTCCGTG GCCGAGAGTA GCATCAACAA CATTAACATG ATATTCCTGT 13383 ACCTGTAAGA AAGTATATCT TGATAGGTAG TTGATTTGCA AGTAClGTGA AAAGGTATCC TATGGAAGAT GTAAAAATAT CTTCATAACA TTATTTGAGA 13483 GGCCAATTTT GGTATCCAAA AATTTCTTAC TTTTRAATTA AGCAACAGAA GTTAGTTAAC AAGTTATTCT ACTCTAGTTT CAGATTTTTT ATGTAGTGTT

13583 TTTGTTTTGT TTGTA TGA GATGATTGAC AAATTGACAT TGACATTCAA CCGTACCCGC CMGCTGTCA TCACAAAAGA GTTGATTGRR ATTATCTCTG

Exon1

S M S I Y D D I D A D V L Q N Y Q E Y N L A N

I I Y Y S L K E S T T S L Q S A K M T A M D N A S K N A

E x o n 8

13683 GTGCTGC GC TC TGAGTA ATTGTAGATT G'ICGCCTTCA GAGAATTTTT TTTCCTCTTG CTGTGTCTGC TTGTTTGGAT GCTTAAAAGT TTTTCGTTTT

13783 TGTCTTTTGA AGTTGAAACT CATTGCTGAA ACTTGCAKC TAACTCT'IAG GCTATAAAGA TGTGGTAAAA ATGGGACCTC TTTGCTTTAT TTTTATTGTT 13883 TCAGCTGGAA ACACTCGGGC TTAGTTTTCG GTTTTCTAAA GGATTAGGAA TGGGATGTGG ATAATTCAGT ATCATTTATG AAATCTTTTT TTGGAATTTA 13983 AACAATTGTA AGCC- TCTGAGTTAC ACGGCTAGTT TCACATTTGG TTTTACATAA ATCTATAAAC CATTGCATGT TGGGAAGGTC CAGGAATTTT

r? E M I D K L T L T F N R T R Q A V I T K E L I E I I S

quence. Figs. 1 and 2 show the structural organization of the gene and the cDNA cloning strategy, respectively.

Sequencing analysis of the genomic locus confirmed on both entire strands revealed that the human ATP synthase y-subunit gene was approximately 23 kbp long and contained 10 exons (Fig. 1) whose length ranged from 37 to 205 bp. The introns ranged in length from 111 bp to 6.9 kbp, with the longest being located in the N-terminal presequence of the gene. The intronlexon boundaries are shown in Table I.

Findings are consistent with the consensus sequence for splice donor and acceptor sites (34), with the exception of the splice donor site of intron 5, which was GC, not the consensus GT. The alteration of GT to GC in the splice donor site of exon 6 was consistent with that in the bovine gene (11). In a novel exon 9, an AG/GT rule was conserved. Comparison of part of the bovine genome sequence showed that the counterpart to the human exon 9 was located in the last intron of the bovine ATP synthase y-subunit gene. The intronlexon boundaries

ATP Synthase y-Subunit Gene and Tissue-specific Splicing 24955 16383 AGACCATCCT GCCTAACTCG GTGAAACCCC G'I'CTCTACTA AAAATACAM AAATTAGCTG GCCGTGGTGG TGGGCGCCTG TAGTCCCAGC TACTATGGAG 16483 CCTCAGGCAC CAGMTGCCA TCMCCTGGC ACGCCGAGCT TGCACXXGC CGAGATTGTC CCACTGCACT CCAGCCTGGG CAAAAGAGCA AGACTCCGTC 16583 TCAAMAAAA AAMAAMAA MCAARTGCA CGAAACC?Ar ? ! A A M C ! rM CASAAACAGC TCGGTCCATT GGCTCATGCT TGTAATCCCA GTATTTTGGG 16683 AGACCGAAGC GAG7GGATTC CTTCAGCCCA GCAC7'I'CAAG ACAAGCL'TGA CCMCATACC AAGACCCCAT CTCTACMAA ATGAAMAAT TAGCCAGGCC 16783 TGGTGGCATC TCCCTGTTGT CCTACCTACT CACCAAA'rTG AGGCACGACG A'TTGCTTGAG CCCAGGAGTT TTAGCCTCCA GTGAGCTATG ATCGTGCCAC 16883 TGCACAAAAC CAAGMTTTA GAMAAGGAA TCCACATAAT TGGAMTTGA GTATTAACTT TTTGCTTCCT AGGAAGCTAG TCTMATCCT TATACAMAA 16983 CTTACCTATG CAGTGCCAAG GTTCGATTGC TTGTGMXG CITTGCATGC TCTGTAACCT ATCAGTTATT CTGAATCMG ACTTGAATAT TTAMCATAC 17083 TTGCAMAGA AAAAGAACM AAMCAMAT MACATATTT GGCTAT'TTAG TAATATAATT AAGMCATGA GTMACCATA GTATTCCAGG TCCMGTATT 17183 AGTTCCCACC CTAAAAACTA AATATGGCCA GGCCCAGTGC CTCATCCCTG TAATCCCAGC ACTTTGGGAG GCCGAGGCGG GTGGATCACC TGACGTCAGG 17283 AGTTCCAGAC CAGCCTGGCC AACATGGCAA MCCCCGTCT CTAC'rAAAM AGTACAAMA TTACCTGGGC ATGGTGCCGG GCACCTGTAA TCCCAGCTAC 17383 TCGGGAGGCT GAGGCAGGAG AATGAC'rTGA ACCCAGGAGG AGGT?GCAGT GAGCTGAGAT CACCCCACTG CACTCCAGCC TGGGCAACAG AACGAGACTC 17483 CATCTCCAAA AAMAAAMA MCC'TC'TAAA TATA??G;<:':' '."AA:.T;ATAG AAMATTGTA AYT'TAMTTA CATAT'TCATA GTCTTGCC'TA CTTGCATGAG 17583 C A T G T T T T f A T l G C f T C f M CC'TGCATTGA MTCTATCAC TT!TTATAAC AGTCATTGAC TCTGTTTCAA AGGAATCTTG TCGTAAGTAT TTTTGCATGA 17683 TGTACTCAAG CTATCAAC?"? TAACAATTGA CTTTT'TT'TCA T'TGCMAGCA TAXTGATTA ACTAGCATGG ATTGCTTCTT CACCTGCTTG TTTTGTGTTT

Exon9 17783 GTCTTTTTCT TCTAATATM TAACCA L A TTAATGAAAA TCAAGTTCCA TCCTCAGACA AGA TAAAG TTCACACATT CTTCCCATGT CTGTTCAGAA

D ... 17883 AAGAAACCTA TTCAGATATA ACTTAAGCTA ATTTAGTAAI' CC'TATAATTC TCCTCTGAGA CATTTAGATG GATGGGCCAC TTCACAGTAG CAGCCAACTC 17983 ACGGGAGCAC CTGGGCAACG CTAGTGCACT TPTTTGTACT CACTCTCI'TG ATCATAGAAA ATGTAGATAT AAGGMATAA ACTTTGAGGA TAGAGAATTA 18083 ACATTATTTT GAGATATTAT ATATACGTTG TGAACAGTCC TGGTTCTCAC ACTTTATGGT CTCTGGACCC CTTAATGTCT GATTCATGTA GCACAAGCCA 18183 GCTAGATTTT CATCTCTCTC TATTCATTTT GTTCTGATGT CATGGATCA'T GTCCCCTCTG GAAAACTCTA CTGTATACTC GAGMTGAGA ATATAACAGG 18283 CAAAATAACA TTATCATGAA AATACTTTTG ACCTCATGAA CCCCATCAM GGTTCCCCAG ACCAAAATTT TAGAATCACT GGTATAGGGT AACACTTTAT 18383 TGTGTAMTT CAGTTCTCTG TACCCCACTT MATATCI'AT TA'?r*ATCICT TGACATTATT TTCCCAAAAA ATGCTGTTTG ATTTCTTACT TGTTCTGTAC

18483 TTTGTTTTTC A TAAAGAA GGAAAATTCA CCCACTTGAT TTTGn'7TTA GCTI'ACTGCT GCCTTTCTCC GAAGAAACTG TTCCTCCATT ATTTGAATTA Exon 10

18583 CTGAAGACAG CAAGATATTT GTAAATTATC T T A A W CAACTTAA*ATCATT GTTTTTCT TATATAAGAA CAATAGATAT AGTTTTTGM ...

18683 ATGAGATGAT ACTAAAACAT TTMAAATAT TAATATGCTA CTATTAAAAT TTTTTAGTAG AAGACAATTT AAGAGGCTCT TTTGATTTGG TAGTGCTCCA 18783 CTGACCTTAA AATGTCTAGC CTC'I'ATCCGC ACAACW'AGA GCGMAGGGC TGCAGCCACA CCCTC

FIG. 3"continued

A B "

1 2 3 4 5 6 kbP

20 - ~ 1 3 . 5 kbp

b j .. C11.0 kbp 7.7 - 6.2 - 4.2 -

.-

IC

3.5 - 2.7 -

rL e 2.5 kbp

FIG. 4. Hybridization analysis of human DNA with cDNA probe for the 'y-subunit of human ATP synthase. Samples of DNA from two healthy males ( A and B ) were digested with restriction endonucleases. The fractionated digests were hybridized with a probe corresponding to exons 2, 3, and 4, under conditions described under "Materials and Methods." Human DNAs were digested with Sac1 (lanes I and 4 ) , HindIII (lanes 2 and 5), and EcoRI (lanes 3 and 6). Marker positions (EcoT141-digested fragments of bacteriophage X DNA) are indicated along the left side of the gel.

of the proposed bovine exon corresponding to human exon 9 were also consistent with the consensus sequence. Nine Alu repetitive sequences were present in the human gene, and are underlined in Fig. 3.

The sequence data of the 3-kbp long EcoRI fragment sub- cloned from the clone XHATPG 2 showed that it was the other allele carrying the EcoRI polymorphism, because at the nucleotide level, exons 2 and 3 were identical with those from XHATPG 23. The intron sequence of XHATPG2 were mostly homologous with that of XHATPG23. A new EcoRI site was located in intron 3 (Fig. 1). This allele lacked a direct repeat 17 bp long, that was present in intron 2 a t nucleotides 7521 to 7554 (Fig. 3).

Examination of the gene organization and the cDNA se- quence for the human ATP synthase y-subunit from HeLa and HT29 cells showed that exons 1 and 2 encoded the N- terminal presequence; exon 2, a portion of the N-terminal end

of the mature protein; and exons 7 and 8, the remaining highly conserved C-terminal region of the mature protein of about 50 amino acids. Exon 10 carried the two polyadenylation signal sequences (Fig. 2).

Gene Structure of the 5'- Upstream Region of ATP Synthase y-Subunit Gene-The nucleotide sequence upstream of the human ATP synthase y-subunit gene contains several struc- tural features which might be important in transcriptional regulation. From the sequence of the 5"noncoding region of HeLa cDNA, the transcription starting site of the human Fl y-subunit gene was a t least 31 bp upstream from the AUG initiation codon. The sequence upstream from the AUG ini- tiation codon contained no TATA box (35), but included other potential promoter sequences, encompassing several Spl binding sites (36) and CCAAT boxes (37) (Fig. 3). The consensus Apl transcription factor binding site (38) was found at -3318 and -3467. Putative AP2 and AP3 responsive elements (39), CCAAT/enhancer binding protein binding ele- ment (40), and serum responsive element (F-ACT1 binding site) (41) were also found in the 5'-upstream region of the human F1 y-subunit gene. As potential sequences important for the coordinational activation of some nuclear genes en- coding mitochondrial proteins, Mt3 (42, 43) and a similar sequence to the NRFl consensus motif (18) were found a t -900 and -43, respectively. The NRF1-like sequence had one nucleotide substitution with t/cGCGCAt/cGCGCa/g to - GGCGCATGCGCG.

Southern' Blot Analysis of Human Genomic DNA-The genomic DNAs derived from the peripheral leukocytes of two healthy Japanese males (A and B) were digested with SacI, HindIII, or EcoRI; Southern blot hybridization was then performed using as a probe the part of the human ATP synthase y-subunit cDNA that corresponded to exons 2, 3, and 4. The results are shown in Fig. 4. Two bands, 11 and 8 kbp, were detected with SacI or HindIII digestion (Fig. 4, lanes 1, 2, 4, and 5 ) . The band 11-kbp long was identical to the functional gene from the analysis of the genomic locus (Fig. 1). When the DNA derived from individual A was digested with EcoRI, three bands appeared (Fig. 4, lane 3): the bands 11 and 2.5 kbp long were identical to the functional gene (see Fig. 1); the other band 5-kbp long was determined to be a processed pseudogene according to the sequence data of the 5-kbp EcoRI-EcoRI fragment subcloned from the ge- nomic clone XHATPG1.' Thus, the 5-kbp band from Southern

* C. Matsuda, H. Endo, S. Ohta, and Y. Kagawa, unpublished data.

24956 ATP Synthase y-Subunit Gene and Tissue-specific Splicing 154 bp

FIG. 5. Alternative splicing of exon 9 in heart and liver. A, nucleo- tide sequences at the intron/exon bound- Liver cDNA I aries around exons 8, 9, and 10 of the human ATP synthase y-subunit gene are shown. The corresponding partial nu- cleotide sequence and predicted amino acid sequences of the resultant alterna- EXON 10 tive mRNAs and proteins are also shown. The size of the PCR products including or excluding the alternatively spliced exon 9 is indicated. B, ethidium bromide-stained 3% aearose eel of the Heart cDNA I - - -CT~GTAA-- - ”

A. I 1 .-L c

Leu m s t o p - - - C T [ G G A ~ A A - - - I I AAAAAA

c .._ ..... GTAA - - - ”” ...... ,

IAAAAAA + Leu Stop I I

117 bp

B. C.

actual PCR fragments ampliked from the heart and liver cDNAs. Sequence of the primers for human tissues is given under “Materials and Methods.” C, the same experiment was performed in bo- vine tissue using a pair of primers, the forward primer (5”AGAGCATGAG- TATCTATGATGACA-3’, correspond- ing to the bovine cDNA a t nucleotides 695-718) and the reverse primer (5‘-

3’, at the nucleotides 999-1020). The size of PCR products was determined by 3% agarose gel electrophoresis.

ATGGACCAATAAATTCTTCTGAC-

HUMAN 10000 12000 14000 16000 10000 20000 22888

. . . . . . .

FIG. 6. Harr plots for comparing of the human ATP syn- thase y-subunit gene with the bovine one. The comparison was made using the computer program GENETYX (Software Develop- ment Co., Ltd. Tokyo). The scale on each axis is in nucleotide base pairs. Each dot represents 15 matching nucleotides out of 20.

Brain cortex Cerebellar cortex Thyroid Liver Spleen Pancreas Kidney Testis Heart Skeletal muscle Intercostal muscle Diaphragm Aorta Stomach Intestine Skin

++ ++ ++ ++ ++ ++ ++ ++ ++ ++

+ ++ ++ ++

f f f f f f rt f f f ++ ++ ++ ++ ++ + + +

hybridization analysis was thought to be the pseudogene. In subject B, other bands 13.5 and 15 kbp long appeared follow- ing digestion with EcoRI (Fig. 4, lune 6). From the character- ization of the genomic locus, the band 13-kbp long was iden- tical to the other allele of the functional gene carrying EcoRI polymorphism (see Fig. 1). The last band 15 kbp long seemed to be the other allele of the pseudogene that carried EcoRI polymorphism, because digestion with Hind111 or Sac1 iden- tified the pseudogene as a single band 8 kbp long. These results indicated that two different genes had been isolated one was the functional gene which had EcoRI polymorphism, while the second gene was the processed pseudogene which also seemed to exhibit EcoRI polymorphism.

Detection of Type of ATP Synthase y-Subunit mRNA-We determined the nucleotide sequence of the HeLa or HT29 cDNA obtained by DNA amplification. Comparison with the bovine heart cDNA sequence (11) revealed a striking differ- ence in the human cDNA from HeLa cells: the 37-nucleotide

FIG. 7. Tissue distribution of transcripts of alternative splicing of exon 9. Each 0.5 pg of poly(A)’ RNA from various tissues was subjected to RT-PCR analysis. These products were visualized in 3% agarose gel electrophoresis by ethidium bromide staining. In the quantitative analysis of PCR products, f implies faint; +, moderate; and ++, strong visible intensity.

insertion at the 3”region of cDNA, as exon 9. To investigate the presence of isoforms of the ATP synthase

y-subunit mRNA, we sought to amplify the y-subunit mRNA from the human heart and liver using a sense primer in exon 8 and an antisense primer in exon 10. The anticipated PCR fragments from the reactions, 154 bp in length from the liver and 117 bp from the heart, appeared in the ethidium bromide- stained 3% agarose gel (Fig. 5B). Sequencing analysis of each amplified fragment revealed that the mRNA from the human liver, but not the human heart, contained exon 9. Analysis of gene structure and Southern hybridization revealed the func- tional gene to be a single gene. From these results, it was

ATP Synthase y-Subunit Gene and Tissue-specific Splicing 24957

Human liver 2 40 s 2 7 3 FIG. 8. Comparison of deduced amino acid sequences of the C-ter- Human heart 239 s 2 1 2

minal region of y-subunit in human Bovine heart 2 3 9 S 2 7 2 heart and liver, bovine heart (ll), E . coli 254 2 8 1 E. coli (50), B. firmus (61), and B. firmus thermophilic bacterium PS3 (52). 2 5 6 2 8 9

PS3 2 5 3 2 8 6

concluded that there are two isoforms of the y-subunit pro- duced by a tissue-specific alternative splicing. The deduced two y-subunit proteins would be identical except for the presence of a single amino acid, Asp273, at the C terminus, because there were in-frame stop codons in exons 9 and 10 (Fig. 5A).

To examine whether the same alternative splicing event is conserved in bovine tissues, RT-PCR experiments were per- formed around exons 6-8 (11) from the bovine heart and liver. The amplified fragment corresponded to exons 7-10 of the human gene. As shown in Fig. 5C, mRNA from the bovine liver contained the new exon between exons 7 and 8, a sequence was homologous to that of human exon 9. When the bovine genome sequence was compared with our data (Fig. 6), the new exon was identified in intron 8 at nucleotide 8746- 8782 (11) in the bovine gene. The bovine genomic organiza- tion, except its 5' end that have not been reported, closely resembled that seen in humans, including the alternative exon.

Tissue Specificity of Alternative Splicing Patterns of ATP Synthase ?-Subunit mRNA in Human Tissues-Several hu- man tissues were analyzed for the diversity of exon 9 in the y-subunit mRNA by the RT-PCR method (Fig. 7). Either the H (heart) type or the L (liver) type mRNA were expressed in many tissues. The cerebrum and cerebellar cortex, thyroid, liver, spleen, pancreas, kidney, and testis mainly expressed the L type mRNA involving exon 9. However, the heart, skeletal muscle, intercostal muscle, and diaphragm, which lacked exon 9, mainly expressed the H type mRNA. The distribution of the pattern of expression in these tissues appeared very clear. Both types of mRNA were detected in the aorta, stomach, small intestine, and skin. It seemed that the H type mRNA was specifically expressed in muscle, which requires a rapid supply of energy, while the L type mRNA appeared to be expressed ubiquitously in various tissues.

DISCUSSION

The complete structure of the gene for the mammalian ATP synthase y-subunit was determined in this study. Its characterization revealed that it spanned approximately 23 kbp and contained 10 exons, with notable features being the presence of a 6.9-kbp intron in the N-terminal presequence, and of exon 9 for alternative splicing. This structural organi- zation resembles that for the partial bovine ATP synthase y- subunit gene thus far described (11). While the reported bovine genomic locus lacked the sequence for 5"untranslated region and part of the N-terminal presequence, we showed that the human gene contained the exon 1 coding 5'41ntran.9- lated region and part of the N-terminal presequence and intron 1 of 6.9 kbp in length. The intron/exon boundary of the human gene conserved an AG/GT rule except for the acceptor site of intron 5 , which was a GT-to-GC substitution. A GT-to-GC substitution at the 5"splice site has been var- iously reported in the genes for human acetylcholine receptor (44), human superoxide dismutase (45), and chicken a-globin (46). A replacement on the 5"boundary site of exon 5 was shown in the bovine F1 y-subunit gene, but it was not related

to alternative splicing. Our results of genomic DNA hybridi- zation and sequencing of genomic clones were consistent with the existence of a single copy gene that carried EcoRI poly- morphism. The presence of two alleles on an EcoRI polymor- phism in two males suggested that the human F1 y-subunit genes were located on autosomal chromosomes. Another proc- essed pseudogene was observed by sequencing analysis.? A similar result of bovine genomic DNA hybridization has been reported (ll), and suggests the presence of a single functional gene and a single pseudogene in cows.

We also showed the existence of tissue-specific isoforms of the ATP synthase y-subunit that were produced by alterna- tive splicing of exon 9. Comparison of the deduced amino acid sequences of the two isoforms showed a similarity except for the addition of Asp273 at the C terminus when the mRNA chose exon 9. Two subclasses of human tissue were clearly determined by the expression of either L type or H type mRNA. When the pattern of expression of the isoforms in various human tissues were analyzed by RT-PCR, three pat- terns were clearly observed. In the heart, diaphragm, inter- costal muscle, and skeletal muscle, most of the expressed mRNA lacked exon 9 and the protein carried no Asp at the end. In the cerebrum, cerebellum, liver, pancreas, spleen, kidney, thyroid, and testis, the mRNA with the insertion of exon 9 was mainly expressed. The skin, aortic wall, stomach, and small intestine expressed both mRNA transcripts. Of the latter four tissues, three contained smooth muscle, and all four tissues contained various kinds of cells. However, our unpublished data showed that human fibroblasts and HT1080, a cell line derived from human fibrosarcoma, ex- pressed both types of mRNA for the F1 y-subunit, even in a clonal cell. Although the distribution of the y-subunit mRNA species clearly differed with the tissues, the function of the F1 y-subunit isoforms was not clear.

F1 is a catalytic portion of FoF1-ATP synthase whose cata- lytic active domain is located either on @-subunit or at (Y-@ subunit interfaces. In a thermophilic bacterium PS3, even the a-p complex showed ATPase activity, although the activity of the a$-y complex was 4-fold higher than that of the a-p complex (47-49). These results indicated that the y-subunit of FI was not necessary for catalysis, but rather for regulation during ATP synthesis. We found that 92% of the amino acid residues of the human y-subunit was identical with that of COWS. Furthermore, when we compared those residues with other amino acid sequences of the y-subunit from prokaryotes, such as E. coli (50), Bacillus firmus (51), and thermophilic bacterium PS3 (52), the conserved sequence was found in the C-terminal region. Fig. 8 shows the alignment of the C- terminal structures of the y-subunit proteins from several species and tissues. This region was previously reported to be important for the function of y-subunit as determined by the analysis of site-directed mutagenesis (53). Interestingly, there is a polar amino acid, at the end of the conserved amino acid sequence, for example, D(Asp) and E(Glu), in B. firmus and in the human liver (Fig. 8).

Tissue-specific isoforms have been reported in an energy production system of mitochondrial inner membrane. Mam-

ATP Synthase y-Subunit Gene and Tissue-specific Splicing

malian cytochrome c oxidase, part of the electron transport system, is a 13-subunit polypeptide complex that contains 10 subunits coded by the nuclear genomes and 3 by the mito- chondrial DNA (26). Three of these nuclear-coded subunits (subunits VIa, VIIa, and VIII) generally exist in one of two isoforms: a constitutive (L) isoform or a skeletal muscle/ heart-specific (H) isoform (27-29). Although these minor subunits are thought to play a regulatory role, their function is not known. Interestingly, the patterns of expression of the ATP synthase y-subunit isoforms resembles those of cyto- chrome c oxidase isoforms. F1 y-subunit isoforms are gener- ated by alternative splicing from a single gene, whereas cyto- chrome c oxidase isoforms are encoded by multiple nuclear genes. Transcription of genes for heart/muscle isoforms of cytochrome c oxidase subunits is thought to be regulated by muscle-specific transcription factors such as My0 D and oth- ers, from the analysis of the 5"upstream region of the genes for these subunits (28). At least in the mitochondrial oxida- tive-phosphorylation system, the post-transcriptional regula- tion of the alternative splicing for F1 y-isoforms seemed to coordinate with the muscle-specific transcriptional regulation of multiple genes for cytochrome c oxidase isoforms.

The steady-state levels of expression of the genes coding F1 subunits have been reported in various tissues. A high level of expression in the heart and skeletal muscle, with lower levels in the liver, cerebral cortex, and other tissues have been observed for the @-subunit of the human Fl-ATP synthase (16) and for the a-subunit of the bovine F1-ATP synthase (10). A similar pattern of expression of the human y-subunit gene was detected by RNA blot analysis (data not shown). Some degree of coordination of the tissue-specific gene expres- sion for the subunits of FI-ATP synthase complex seems likely. As a functional NRFl element is reported in the 5'- noncoding region of the bovine ATP synthase y-subunit gene (54) and the human gene has a NRF1-like sequence, NRFl presents a possible mechanism for the coordinated activation of gene. An analysis of the coordination system is now in progress.

Recently, we have determined the primary structure of the C-terminal peptide fragments of the two isoforms of the bovine Fl y-subunit, so that the liver type isoform had an additional aspartate residue at the C terminus, not present in the heart type one (55) .

In conclusion, we determined the complete structure of the human ATP synthase y-subunit gene and its two isoforms generated by muscle-specific alternative splicing.

Acknowledgments-We thank Dr. H. Nojima for the gift of cDNA libraries, Dr. S. Miyabayashi for the gift of a part of human tissues, and S. Akiyama for the human genomic library and his genomic DNA. We also thank M. Hoshino for her expert secretarial assistance.

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