217

Biochimica et Biophysica Acta, 1131 (1992) 217-219 © 1992 Elsevier Science Publishers B.V. All rights reserved 0167-4781/92/$05.00

BBAEXP 90361

Short Sequence-Paper

Upstream region of a genomic gene for human mitochondrial transcription factor 1 Kaoru Tominaga, Shuichi Akiyama, Yasuo Kagawa and Shigeo Ohta Department of Biochemistry, Jichi Medical School, Minamikawachi-machi, Tochigi-ken (Japan) (Received 26 March 1992)

Key words: Transcriptional factor; Mitochondrion; Genomic gene; Upstream region; (Human)

A genomic gene for human mitochondrial transcription factor 1 was cloned from a human genomic library and its 5' flanking region was sequenced. No typical TATA and three consensus sequences for potential Spl binding site were found in its 5' flanking region of 2 kilobase pairs. There were, at least, four common sequences among some nuclear genes for mitochondriarelated proteins.

The mitochondrion has its own genome and genetic expression system [1]. The human mitochondrial genome encodes 13 polypeptides of complexes of the respiratory chain and ATP synthase [2]. Most mitochondrial proteins are encoded by nuclear genes including all protein factors involved in the mitochondrial genetic expression system. Mitochondrial transcription is regulated depending on the cellular conditions. For example, it is enhanced by immortalization and repressed by anaerobic conditions [3-5]. Mitochondrial transcription factor 1 (mtTF1) works as a transcriptional activator by binding to both the lightand heavy-strand promoters on mitochondrial DNA [6,7]. cDNA encoding human mtTF1 has been cloned and sequenced [8]. In order to investigate the regulatory mechanism of the mitochondrial transcription, it is essential to know how the regulatory mtTF1 gene is expressed. Here, we cloned a genomic gene for the human mtTF1 and sequenced its 5' upstream region. In order to prepare a genomic gene library, total DNA was isolated from peripheral blood cells of a Japanese man, digested partially with a restriction endonuclease MboI and then ligated into the B a m H I site of A DASH II (Stratagene). For hybridization probes for cloning the

Correspondence to: S. Ohta, Department of Biochemistry, Jichi Medical School, Yakushiji, Minamikawachi-machi, Tochigi-ken, 32904 Japan. The sequence data in this paper have been submitted to the EMBL/Genbank Data Libraries under the accession number X64269.

gene, DNA fragments corresponding to the 5' flanking region as well as the coding region of m t T F I - c D N A were obtained by the polymerase chain reaction (PCR) method coupled to reverse transcriptase [9]. The first strand of the cDNAs of HeLa cells was synthesized using oligo(dT) as a primer, and then subjected to the PCR reaction. The PCR-primers used are as follows; for fragment A, 5 ' - T C G C T A G T G G C G G G C A T G A T (from cDNA-nucleotide number 3 to 22) and 3'C G C T A C C G C A A A G A G G C T T C (from 130 to 149) and for fragment B, 5 ' - A T G G C G T T T C T C C G A A G C A T G T (from 133 to 154) and 3'-GCTFTTATACCACGACTCCTCAC (from 847 to 869). Out of 1.2 • l06 independent recombinants, 14 clones positive against both fragments A and B were isolated. Three of these clones were distinguished from the others by digesting the phage DNA with HaeIII or MboI. A HaelII fragment (1.5 kilobase pairs (kbp)) was hybridized with both cDNA fragments from nucleotide number 133 to 508 and from 574 to 869, suggesting that they are intron-less genes. In addition, the DNAs from the three clones were not hybridized with an oligonucleotide (from nucleotide number 3 to 22), while the other 11 clone DNAs were. Therefore, the three clones were excluded for selection of the bona fide genes. It was consistent that the other 11 clones overlapped each other as judged by Soutern hybridization. A clone of them was picked up and an E c o R I fragment (5 kbp) was subcloned into pBluescript SK + (Stratagene). Since its HindlII-EcoRI fragment was hybridized with fragments for the 5' flanking region and 5' region of the coding one (from cDNA nucleotide number 133 to

218 TTGTCAGTAGCATGAAATCTGCCAATGGTGGGAGTATTTACACCATAGAACTCAGCAAACAC CACAAATTGGGGCTTTCTTTTTTGGAGAGCTATTTACCAGCATATTACTGGCCTGAAC -2147 AAAAGCAACAGCAATGGGACTAGAGAGAAGGAAATACATACTAGAGGGATGAATGAGGCAGAATCAGCAATACTCATCATCAGTTGGTTAATGGCAGGTGGAGTGTCAATGGGGAGATTC

-2027

TAAAATGATTTATCAAGTTATTGGCTCAGATGATTAAAGAGGAAAACATGTGCATCTTACCAAGAGAAGGACTTCAAGATGGGT~GATAGTGGATTTTGTTCAGGGAATGTTGAAAT

-i 907

TGAAGTAcTTGTGGGACATGATGGTGAAAATGTACAGAAGACAGTGGTTATGTGGATACGGAA•TCAAAGGAGAGGACTGGGTTCAATATGGCAGTCATGAGG•TATAACTAATAGCTGA

-1787

AG T C A T G G G A C T A G A G A A C T T T A C A A C A A A G G G A C c • T G G G A A A G G C T G C A G T A A G T C T T C A T G T T T T A T A A A G T A A T T A T A T A C C A T G T C T T T T T G T T A A A A G A c A G T T T T A G T G G A T A

-1667

AAGTTATGTTTCTTTGTAAATTCTGCTTTTAGAAGTAAAATTATCACTG•AATGTAAATGAGTTTTAAGGAAGATTGGTATCTGATGAAAAATGTTATGGATTTGATTTTTAAGGT•TTT

-I 547

ACAGCAAGATTTATACAAATGAAAGTCGAAAACAACAAAAAGTTTAATATCCAAAAGAAGTTTATGTCAAACATTTGAAAGAACAAAAACATTCTAGAA•AAAAAGGGAAACAAATTATT

-1427

T T A G A A A T C A A C T T A A A T G T A A C T T C T G T C T G A G C C C C A T G T T G T A A T T A T T T GCTAC T T T G C C T C A C C T T ~ G G G G C T G ~ ( ~ G G C G G G ~ ( ~ - - ' ~ ' - ~ C A C A A C A C T G T G

TCTTTTAT

-1307

CTATGTTTAGGGC G TTGTACATAGTATATACTCAGGAAAAAAACCTGTTTATTCCATGCCAGAATGGGAGGTTTGAACAGATGACTTGGAAGGTCCCTTTCAACCCTGGGAGTCTATAAA

-1187

AGGGAAGTGGTTATTACCGCTTAAAAGAAC, A A G T A G G C TTAGTC C T A C T T C C A G A G A G C C A A T G T T T T C A T A G T T A C A G A A A G G T A A T A G A C T G T C A G C T C A A T A T G A C A C T G G T A A G A A

-1067

TTTTCTAACAATTACGGCTAT•CAACAGAAGAAAGGCTGTCTCAGAAGGTGGTTAGCTCTGAAACAACTGGTATTTAAGGGCTGGAAGGACCCTTAGATATCTTTAGGTCTTCCTAATCT

- 947

TTTT•ATG••ATTG•CTATACAGAAAATGGTATTTCTATAACACAAATAAAAAAATTATACAGCAATTTTTGACACATCAATGTCTCAAGGCATACTTGTCACACGTTCTCAGGGCTTTG

- 827

G•TGGGAAAACTGATTTAGTCTAACAT•CT•GTTTGCAGAGGGGGAAACTGAAT•TTAACGAGGGAATGCAATATTC•CAAGTCAAGACGTTAATGTTAATACTGGAACTAGAAGAATTG

- 707

AGACTAGCGACTGTGGACAACTAGCCAGTTTCCTCTGCTCAAATTAAGCAAG•TGGAGGCCAGACTGAAAGACTGCAGTAGATGGTAGAATAAACAGTcTAAAGCTATATTGCTTTAAAT

- 587

GTGGTCTCAAGAC~ACCGA~ATTAGAA~TTGTTAAATTCTGGGGAACTCGCTAAAATGAAGATTAATGGG~CCCACATAGACACACGGAATTAAGCTCTGCGGTAAGGC~TTGGAATCTG - 467 CATTT T T A G G T T T G C G A A T C C C C G C C T C T C C C G T TACTAT TTC TGAACT CCGAGCT CCAGC CCTGGCT T G A A C T G A G A C G C T C C G C T G G G C G C G C A G C A G C C G C C G A T C G G A C C T C G G G G

- 347

TC•TGGATGCAGGACTGTCTGTTACGTACAGCCCTTGTGACCGTCACGGGCGGATACCGGCCAACGC•GGGTTGGGGTGAGGCCGCCGCCGCGGTCCCTCCATCACCCTCCTGGCCCGGC

- 227

AGAGGAACCCACTG•TCCGGGC•GC•GGGGACAGAGGTGGCTCAA•AGAGCCGCCTCGAAGCcAGAGCCCTCCGCAGGCTAGAGGATTGCGGTTTCCCTTCATCTCCGCGGCTCTTATTC

- 107

-I+i CT C C C C C G C G A G G C C G C C C A C C G G G G T A C G C T C T C C C G C G C CTGCGC CAATTCCG CCCC GCC CC GCC CCC ATCTACC GAC C G G A T G T T A G C A G A T T T C C C A T A G T G ~ . ~ T A G T G G C

G

+

14

~CATC~T1~CACA~GCCGGAGGGTCC~ACGCC.4~GTTCCAG'I~GTGATTGCTGG'AGTTGTGTATT~2CAGGAGG~TCTCCGAGATT~GGTCC~GTCA~`.~GC + 134

GG~GTTTCTCCGAAGCATGTGGGGCGTG~TGACTG~C~TGGGAAGGTCTGGAGCAGAG~TGTGCA~CGGCTGTGGAAGTCGACTGC~T~CC~T~A~TAGG~CCGC

TTGCCT GTGCC

+ 254

tA~aPheLeuAr~SerMetT~G~Va~LeuThrA~aLeuG~Ar~Ser~yA~aG~uLeuCYsThrG~YC~sG~SerAr@L~uAr@SerPr~PheS~ CTAGGGGCAGCAGGGCCCAGGACGTCCCGGGGTTGGAATGTAGACCCTATCCTTCACTTTCTGCCCCTCCTAGGAGTTCAGAGTCACCCTGGTT•TTTGATT•AGACCGCGACCTTGCcA

+ 374

AGGGGACGGTGC-CCTTGAGACCAGGCCTTTAT TTACTCTC T T A A T A C C C CGTACC TTCTTGC T A C C C T C C A C T C G C T T C T C C C A T C C C T C C T C A C T G T C C A G C T T T G G A T C C T T T C CGCC

+ 494

TCCTCTGCGAATTC

Fig. 1. Nucleotide sequence of a part of a genomic gene of the human mitochondrial transcription factor h This sequence corresponds to the 5' flanking region, the first exon and a part of the first intron. The amino acids encoded in the first exon are added in a box. The transcription start site was numbered as + 1 according to Marisi et al. [8]. Consensus sequences for a potential Spl binding site are shown by open boxes, and GC-rich sequences are underlined.

508), it was s e q u e n c e d on b o t h s t r a n d s by the u n i d i r e c tional d e l e t i o n m e t h o d [10]. T h e 5' flanking n o n c o d i n g s e q u e n c e was c o m p l e t e l y i d e n t i c a l with that of e D N A . In o r d e r to c o n f i r m it was a b o n a fide gene, the p r o m o t e r activity was e x a m i n e d as follows. T h e E c o R I f r a g m e n t was c o n n e c t e d to t h e c h l o r a m p h e n i c o l acetylt r a n s f e r a s e ( C A T ) g e n e a n d t h e n t r a n s f e c t e d into H e L a cells. T h e e x p r e s s e d C A T activity was c o m p a r a b l e to that of the A T P synthase /3 subunit gene [17] ( d a t a not shown). T h e r e f o r e , t h e gene s e q u e n c e d h e r e is evident to b e b o n a fide. Fig. 1 shows the n u c l e o t i d e s e q u e n c e for the 5' u p s t r e a m region, the first exon a n d a p a r t of the first intron. T h e t r a n s c r i p t i o n start site was n u m b e r e d as + 1 a c c o r d i n g to Parisi et al. [8]. T h e r e w e r e no typical T A T A a n d t h r e e c o n s e n s u s s e q u e n c e s for p o t e n t i a l S p l b i n d i n g site [11-13] a n d p o i n t e d o u t by boxes in Fig. 1. T h e regions from n u c l e o t i d e n u m b e r - 2 6 6 to

- 2 7 7 a n d from - 107 to - 5 9 are m a r k e d l y rich in G C (82.5% a n d 83.7%, respectively). So far, c o m m o n seq u e n c e s a m o n g s o m e n u c l e a r g e n e s o f the m i t o c h o n drial p r o t e i n s have b e e n r e p o r t e d . H o w e v e r , the seq u e n c e s c o m p l e t e l y m a t c h e d with N R F - 1 [14], M t l , Mt3 o r M t 4 [15] w e r e not f o u n d in this sequence. T h e n u c l e o t i d e s e q u e n c e of the 5' u p s t r e a m region was c o m p a r e d to those of the o t h e r g e n o m i c genes r e p o r t e d in the n u c l e o t i d e s e q u e n c e G e n B a n k d a t a b a s e ( L A S L - G D B ) . Fig. 2 shows 8 or 9 b a s e s m a t c h e d new c o m m o n s e q u e n c e s a m o n g some n u c l e a r genes for the o t h e r m i t o c h o n d r i a l proteins. T h e c o m m o n s e q u e n c e A in Fig. 2 is the c o m p l e m e n t a r y one of PU box ( G A G G A A ) which has b e e n i d e n t i f i e d as the b i n d i n g site of a m a c r o p h a g e and B-cell specific t r a n s c r i p t i o n factor [16]. This s e q u e n c e a n d the o t h e r c o m m o n seq u e n c e s m a y play a role in c o o r d i n a t i n g the n u c l e a r a n d m i t o c h o n d r i a l gene expression.

219

References

A TTCCTCTG ******** Human.mtTFl Human.F1 Beta

-681

C A G ~ T C A A

-665

-82

GTA~TCCTCT~TTAC

-66

Human.PDH Beta

-1463

AATyTCCTCT~TCTT

-i 447

Rat.UCP

-1491

AGGG~TCCTCTG~GTAG

-1475

B TTCTGCTT ******** Human.mtTFl

-1650

T A A ~ T A G A

-1634

Human.ANT-I

-1331

TTC~TCTGCT~CCTG

-1315

-585 -501

CCATTCTGCTTAGGA CAATTCTGCT~AGTT

-568 -485

Human.PDH Beta Human.PDH Beta Rat.UCP

-1270

GGCA~TCTGCTT~CCTC

-1254

C AAGCTGGAG ********* Human.mtTFl Human.Fl Beta Rat.UCP

-1016

-661

AAGC~AGCTGGAG~CCA i | GCCC~AGCTGGAG~GCA

-I000

-645

-757

ACCA~AGCTGGAG~ACT

-740

D TTCCTCCCC Human. mtTFl

-113

Human. ANT-2

-285

CTTA~TCCTCCCC~GCG I ! ACCC~TCCTCCCC~CTC

-269

-97

Rat .UCP

-515

GGAT~TCCTCCCC~CCT

-499

Fig. 2. Common sequences among the genes of the other mitochondria-related proteins. Eight bases-matched sequences were searched against the GenBank nucleotide sequence database. Abbreviations used are as follows: mtTF1, mitochondrial transcription factor 1; F1 Beta; ATP synthase/3 subunit [17]; PDH Beta, pyruvate dehydrogenase/3 subunit [18]; UCP, uncoupling protein [19]; ANT-l, adenine nucleotide translocator-1 [20]; and ANT-2, adenine nucleotide translocator-2 [21]. It should be noted that A is identical to the complementary sequence of the PU box which has been found as the binding site of a macrophage and B-cell specific transcription factor [16].

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