Biochimica et BiophysicaActa, 1089 (1991) 283-285

283

© 1991 Elsevier Science Publishers B.V. 0167-4781/91/$03.50 ADONIS 0167478191001657 BBAEXP 90237

Short Sequence-Paper

Nucleotide sequence of the last exon of the gene for human cytochrome c oxidase subunit VIb and its flanking regions J a n - W i l l e m T a a n m a n , C o b i S c h r a g e , E v e r t B o k m a , P e t e r R e u v e k a m p *, Etienne Agsteribbe and Hans De Vries Laboratory of PhysiologicalChemistry, Unit~ersityof Groningen, Groningen (TheNetherlands) (Received 22 March 1991)

Key words: Genomic DNA sequence; Alu repetitive element; Cytochrome c oxidase; Mitocbondrial protein; (Human)

A human genomic clone encompassing the last exon of the gene for cytochrome c oxidase subunit Vlb and a human genomic clone containing the most distal end of this gene were characterized. The last exon of the gene codes for the 17 C-terminal amino acid residues of the subunit and the 3' noncodiug region. Downstream from the gene we found a single base difference between the DNA sequences of the two genomic clones. An inverted A/u dimer repeat was identified further downstream.

Eukaryotic cytochrome c oxidase (EC 1.9.3.1) is localized in the mitochondrial inner membrane. It transfers electrons from cytochrome c to molecular oxygen in the terminal reaction of the respiratory chain and converts the flee energy of the electron transport into an electrochemical proton gradient across the inner membrane [1]. The three largest subunits, which appear to form the catalytic core, are encoded by the mitochondrial DNA. In mammals, the enzyme complex consists of ten subunits of nuclear origin in addition to the mitochondrially encoded subunits [2]. Some nuclear-encoded subunits are present as tissue-specific isoforms [3,4]. The function of these nuclear-encoded subunits remains to be established. We are characterizing the human enzyme. The genes encoded by the human mitochondrial DNA have been characterized and sequenced [5], but little is known about the features of the nuclear genes of cytochrome c oxidase subunits. Recently, we isolated a human

The sequence data reported in this paper have been submitted to the EMBL/Genbank and DDBJ Nucleotide Sequence Databases under the accession number X58139. * Present address: Department of Medical Genetics, University of Groningen, Ant. Deusinglaan 4, 9713 AW Groningen, The Netherlands. Correspondence: J.-W. Taanman, Laboratory of Physiological Chemistry, University of Groningen, Bloemsingel 10, 9712 KZ Groningen, The Netherlands.

eDNA specifying the nuclear-encoded subunit Vlb of cytochrome c oxidase [6]. There are probably no tissue-specific isoforms present of this subunit [7]. In a

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Fig. I. Schematic presentation of the cloning and sequencing strategy of cytochromc c oxidase subunit VIb genomic sequences. The upper part of the figure shows the cosmid clone, el3, and bacteriophage clone, GI3; thin lines represent vector sequences, thick lines rep;esent cloned genomic sequences. The lower part of the figure shows a physical map of the last exon of the gene and its flanking regions; the exon is represented as a box with the coding part being shaded, positions and orientation of A/u repeats are indicated with arrows in the map. Arscs~ below the map indicate direction and extent of sequencing; thin arrows indicate sequences derived from cl3-subclones, thick arrows indicate sequences derived from Gl3-suhelones. Restriction enzymes ased for subcloning and seque:ging are denoted as follows: B, BamHi; E, EcoRi H, H/ndlll; K, /0ml; /14, Mbol, P, Pstl, S, Sa/l.

284 ~ATCCATCC

A~CA~CCA

G~CAT~T

CAGCCTCGTT T T T T T T C A C A ~ T G C C T G C T

ATTCCATCTC

70

CCC~TT~

AC~CAGCA

TAGTGTTGAC T T G A ~ T T T T

C~GCAG~

CCCACCCTTG G A G C T C T G T A

140

CAG~ACACA

TCTCCCCATC GACAGCTCTG C A G T ~ G C C T

G~GTTTTCC

TGCCCATTAC T C T C C T C C C A

210

GCA~TCAGC

TTGTTCAG~

CAGTGATTTC TGTCTTTTGG G G T C A C T G C T G A T T C C C C G G C C T C T A G A A T

280

AGA~TTGGC

ACACAGCA~

TACCTGT~A

G~CAAAGTG

AGGAAGATAA

350

G~GTC~TC

CGTTCAGTTT CCCCACTGCG G A G G G A A T A A CACTGTCTTT C C A C A ~ T C A

CAGACTGGGA T D W D

420

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490

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CCA~A~T

GAA~AC

C~TACCCA

G~ATCCCCA

CCCCA~ATC

CTAAATCATG

560

ACTTACC~

T~TA~AC

TCATT~A

AG~CTA

TGCGTG~

CG~CCA~C

AG~GC~GA

630

TGTTTTTATT

700

~AG~CAAT

770

l AGC~CTG

G~TCAGTGC

CC~ACCAGG

GAG~CTTG

A_~GAGTATT TTCTC~TTT t~

TA~TAATTT

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CACGCA~CT

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910

CCA~TC~C

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CCAGACTGGA 1120

G~AG~GT

GCGC~TC~

AGCC~CTGA

CTCCAGTGTT CAAGCGATTC T C C T G C C T C A G C C T C C C G A C

CCTC~CCTC

CC~GTGCT

840

ACA~TC~

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CTCACCACCG C ~ C C T C C G T

CCCAC~C

T C A A G T G A T T CTCCTGCCTC 1190

GTAGC~GA

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AGCCTGGCTA ATTTT~TAT

GACA~T

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GCCA~C~

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CACGAC~CCC A G C C C T A ~ A

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1260

A T G A T C C A C C TGCCTCGGCC 1330 CTTTTCAATA ACAA~TTA

1400

AT~GAGAT ~CT~ATA C ~ A A C A G T A T A T G G C T ~ A C ~ T T C A A A C TAGAATTC 1458 Fig. 2. Nucleotide sequence of the last exon of the gene for cytochrome c oxidase subunit Vlb and its flanking regions. The exon is boxed, the deduced amino acid sequence is show below the exon. The intron/exon boundary (~,), the stop codon (*) and the sequence of the oligodeo~nucleotide used for screening and sequencing (underlined) are indicated. Alu repetitive elements are underlined by arrows. The two h~e~ at nucleotide position 672 indicate a sequence difference between the two genomic clones. The Mbol site (GATC) at nucleotide positions 454-457 represents the 5' end of the cloned genomic insert of G13.

first attempt to isolate the subunit VIb gene [8], we screened under stringent conditions, with the subunit VIb c D N A as a probe, 5 . 1 0 5 plaques of a library (kindly provided by Dr. G.C. Grosveld, Erasmus University, Rotterdam) constructed from 15-20 kb fragments of partially Mbol-digested human genomic DNA, cloned into the bacteriophage EMBL3 [9]. Seven of the nine positive clones obtained from this screening appeared to contain three distinct loci with different processed pseudogenes for subunit Vlb; their sequences have been published elsewhere [8]. Restriction

mapping and Southern blot hybridization indicated that the remaining two positive clones were identical to one another. Fragments that hybridized to the c D N A probe of one of the clones (G13) were subeloned into the plasmid vector p U C I 9 [10] under standard conditions [11] to facilitate sequence analysis. Sequencing was performed by the chain-termination procedure with denatured plasmid templates and using the universal primer for pUC19 as described [8]. The sequencing strategy of G13 is depicted in Fig. 1. Comparison with the subunit Vlb c D N A sequence revealed that G13

285 contained a sequence identical to the 3' end of subunit VIb e D N A . At the 5' end, the sequence of G I 3 was interrupted at a Sall site adjacent to an Mboi site. Since the library was constructed from partially Mbol digested material and since cloned fragments in EMBL3 are flanked by Sail sites [9], it appears that G I 3 only contains the most distal end of the gene. The sequence downstream from the M b o l site is shown in Fig. 2. The sequence does not contain a poly(A)-tract at the 3' end, which is one of the hallmarks of processed pseudogenes. Therefore, we assume that G I 3 represents part of the expressed gene and its 3' flanking region. T o further characterize the subunit Vib gene, we screened a second human genomic library (kindly provided by Dr. L. Blonden, University of Leiden) of 40 kb M b o l - r a n d o m fragments, inserted into the cosmid vector c2RB [12] and amplified in Eschenchia coli 1046 (Rec A - ) . A total of 4 . 1 0 s recombinants on G e n e Screen plus (DuPont) filters were screened with subunit V l b c D N A , labelled with [a-32p]dCTP [13], at 65°C under hybridization conditions described for genomic sequencing [.'4]. To discriminate between clones containing processed pseudogenes and clones containing the expressed gene, duplicate filters were hybridized at 55°C to a 32p-end-labelled [11] synthetic oligodeoxynucleotide corresponding to the sequence of G I 3 just downstream from the sequence identical to the subunit Vlb e D N A sequence (indicated in Fig. 2). The screening with the e D N A yielded 27 positive clones. Only one of these clones (c13) gave a strong hybridization signal with the oligodeoxynucleotide probe. Clone c13 was mapped (Fig. 1) and fragments of interest were suheloned into pUC19 for sequence analysis. The sequencing strategy is shown in Fig. 1. Sequence results are presented in Fig. 2. Comparison with the c D N A sequence of subunit Vlb reveals that clone c13 apparently contains the last exon of the subunit Vlb gene and its flanking regions. The i n t r o n / e x o n junction (nucleotide 406-407, Fig. 2) is consistent with the consensus sequence [15]. The characterized exon contains the coding sequence for 17 of the 85 amino acid residues of subunit Vlb and the 3' noncoding region of the gene. The sequence is in full agreement with that of the previously isolated subunit Vib c D N A [7]. Clone c13 has been used as a probe to

localize the subunit Vlb gene on chromosome 19 band q13.1 [16]. Downstream from the gone we found a single base difference between sequences derived from clone G I 3 and from clone c13 (nucleotide 672, Fig. 2; A in c13, G in G13). This variance was confirmed by sequencing a G13-subclone with the oligodeoxynucleotide used to screen the cosmid library as a primer for better resolution. The base difference may repre,sent an allelic variant. An inverted Alu dimer repeat was found downstream from the gene. Such repeats must statistically be dispersed throughout the human genome at 5 kb intervals [17]. Two of the three processed pseudogenes we isolated previously were interrupted by A/u repetitive elements [8]. Therefore, it appears that cytochrome c oxidase subunit Vlb related genomic sequences are associated with Alu repeats.

References ! Hatefi, Y. (198-';)Annu. Rev. Biochem. 54. 1015-1069. 2 Capaldi, R.A, (1990) Annu. Rev. Bioehem, 59, 569-596. 3 Kadenbach, B., Kuhn-Nentwig, L. and Bilge, U. (1987) Current Topics Bioenerg. 15, !!3-161. 4 Yanamura. W., Zhang, Y.-Z., Takamiya. S. and Capaldi. R.A. (1988) Biochemistry 27. 4909-4914, 5 Anderson, S., Bankier, A.T., Barrell. B.G.. De Bruijn. M.HL, Coulson, A.R,, Drouin. J., Eperon, I.C.. Nicrlich, D.P., Roe, B.A.. Sanger, F.. Schreier. P.H.. Smith, AJ.H., Staden. R. and Young, I.G. (1981) Nature 290, 457-465. 6 Taanman, J.-W., Schrag¢, C., Ponne. N.. eolhuis, P.. De Vries. H. and Agsteribbe, E. (1989) Nucleic ,Acids Res. 17, 1766. 7 Taanman, J.-W.. Sehragc, C, Ponne. N.J., Das, Aft.. Bolhuis. P.A, De Vries, tL and Agstcribbe, E. (1990) CJcn¢ 93. 285-291. 8 Taanman, J.-W.. Schrag¢, C.. Reuvekamp. P., Bijl. J., Hartog, M.. De Vries. H. and Agsteribbe. E. (1991) Gene, in press. 9 FrischauL A.-M., Lehrach, H.. Poustka, A. and Murra% N. (1983) J. Mol. Biol. 170. 827-842. 10 Yanisch-Perron, C., Vieira, J. and Messing, J. (1985) Gene 33, 103-119. II Sambrook. L, Fritsch, E.F. and Maniatis. T. (1989) Molecular Cloning: A Laboratory Manual. Second Edition, Cold Spring Harbor Laboratories. Cold Spring Harbor. 12 Bates. P.F. and Swift, R.A. (1983) Gene 26. 137-146. 13 Feinberg, A.P. and Vogelstein, B. (1983) Anal. Biochem. 132. 6-13. 14 Church, G.M. and Gilbert, W. (1984) Proc. Natl. Acad. SCI.USA 81, 1991-1995. 15 Mount. S.M. (1982) Nucleic Acids Res. 10. 459-472. 16 Taanman. J.-W.. Van der Veen, A.Y., Schrage, C., De Vfics, H. and Buys. C.H,C.M. (1991) Hum. Gpnet.. in press. 17 Schmid. C.W. and Jelinek. W.R. (19821 Science 216. 1065-1070.