Hum Genet (1991) 87:325-327

[ (ETi32gii 9 Springer-Verlag1991

Assignment of the gene coding for human cytochromec oxidase subunit Vlb to chromosome 19, band q13.1, by fluorescence in situ hybridisation Jan-Wiilem Taanman 1, Anneke Y. van der Veen 2, Cobi Schrage 1, Hans de Vries 1, and Charles H. C. M. Buys 2 1Laboratory of PhysiologicalChemistry,Universityof Groningen, Bloemsingel10, NL~9712KZ Groningen, The Netherlands 2Department of Medical Genetics, Universityof Groningen, Ant. Deusinglaan4, NL-9713 AW Groningen, The Netherlands Received January 23, 1991 / Revised February 26, 1991

Summary. A cloned, 40 kb, genomic DNA fragment, containing the last exon of the gene for human cytochrome c oxidase subunit VIb and its flanking sequences, was used as a probe to localize the subunit VIb gene on human metaphase chromosomes. The probe was labelled with Bio-ll-dUTP and detected by fluorescence. Subsequent R-banding indicated that the cytochrome c oxidase subunit Vib gene is localized in band 19q13.1, extending the evidence that the human nuclear genes of cytochrome c oxidase are not clustered.

Introduction Eukaryotic cytochrome c oxidase (EC 1.9.3.1) is integrated in the mitochondrial inner membrane. The enzyme complex transfers electrons from reduced cytochrome c to molecular oxygen and contributes to the establishment of a proton gradient across the inner membrane in the terminal reaction of the respiratory chain (reviewed in Hatefi 1985). Like most complexes of the respiratory chain, cytochrome c oxidase consists of polypeptides of dual genetic origin. In mammals cytochrome c oxidase is composed of three large, catalytic subunits (I, II, and III) encoded on the mitochondrial genome and ten smaller subunits (IV, Va, Vb, Via, Vib, VIc, VIIa, VIIb, VIIc, and VIII) encoded on the nuclear genome (reviewed in Capaldi 1990). The nuclear-encoded subunits are thought to be involved in the regulation of enzyme activity and the assembly of the complex (Kadenbach 1986; Bisson et al. 1987; Poyton et al. 1988). Some nuclear-encoded subunits are present as tissue-specific isoforms (reviewed in Kadenbach et al. 1987; Yamamura et al. 1988). The genes encoded on human mitochondrial DNA have been characterized and sequenced (Anderson et al. 1981), but little is known about the features of the nucleOffprint requests to: J.-W. Taanman

ar genes of the cytochrome c oxidase subunits. For most human nuclear-encoded subunits, eDNA clones have been isolated and sequenced (reviewed in Lomax and Grossman 1989). Some of the genes have been mapped to human chromosomes. The subunit VIII gene has been mapped to 11q12-q13 (Rizzuto et al. 1989) and the subunit IV gene has been mapped to 16q22 (Lomax et al. 1990). Furthermore, a processed pseudogene for subunit IV has been located on chromosome 14 (Lomax et al. 1990). We have isolated a human eDNA clone coding for subunit VIb (Taanman et al. 1989). There are probably no tissue-specific isoforms of this subunit (Taanman et al. 1990). Recently, we isolated and characterized human genomic clones, corresponding to three different genomic regions, containing processed pseudogenes of subunit VIb (Taanman et al. 1991). In addition to the processed pseudogenes, which lack introns, we have isolated an intron-eontaining, cosmid clone and assume that this clone represents the expressed gene of subunit VIb. Because of the presence of processed pseudogenes and the relatively short length of the full-length eDNA (450 bp), the intron-containing cosmid clone has been employed in the physical localization of the cytochrome c oxidase subunit VIb gene to human metaphase chromosomes by non-radioactive in situ hybridization.

Materials and methods Isolation and characterization o f probe

A human genomic library of 40 kb MboI-random fragments, inserted into the cosmid vector c2RB and amplified in Escherichia coli HB101 (Bates and Swift 1983), kindly provided by Dr.L. Blonden (Univ. of Leyden, The Netherlands), was screened with full-length cDNA, specifyingsubunit VIb of human cytochromec oxidase (Taanman et al. 1990), labelled with [u-32p]dCTP (Feinberg and Vogelstein 1983). One hybridizing clone (c13), characterized by DNA sequencing, appeared to contain the last exon of the gene codingfor subunit VIb and part of the last intron, indicat-

326 ing that this clone probably represents the expressed gene. Alu repetitive elements were present in both in the intron and in the sequence downstream from the last exon (manuscript in preparation). D N A of clone c13 was isolated from cultures by centrifugation in a CsCl-ethidium bromide gradient (Sambrook et al. 1989). To investigate the presence of repetitive elements in the genomic clone, a Southern blot of c13 D N A , digested with BamHI, EcoRI, HindIII and PstI, was hybridized to radioactively labelled total human genomic D N A . Southern blotting and hybridization were carried out as described in Taanman et al. (1991). The hybridized blot was exposed to X A R film (Kodak) overnight.

Chromosome preparations Human lymphocyte metaphase spreads were obtained from whole blood cultures in M199 medium. After 3 days of culture, BrdU (5bromodeoxyuridine) was added to a final concentration of 0.1 mM for the last 6 h, and cells were arrested in metaphase by adding colcemid to a final concentration of 50 ng/ml 1.5 h before harvesting the cells. The cells were subjected to hypotonic treatment in 0.075 M KC1 and fixed in a mixture of methanol and acetic acid (3 : 1, v/v).

buffer, pH 5.2, at 87~ for 1-2 min, rinsed with the pH 6.5 buffer, and mounted in antifade medium (CiTi-Fluor Ltd., London) containing propidium iodide to a final concentration of 2 pg/ml. Micrographs were taken using a Leitz Aristoplan-photomicroscope with filter combinations BP450-490/LP15 and BP515-560/LP580 for FITC and propidium iodide, respectivley.

Results and discussion To determine the chromosomal localization of the cytochrome c oxidase subunit VIb gene, we hybridized metaphase chromosome spreads with a biotinylated genomic c l o n e c o n t a i n i n g t h e last e x o n o f t h e c y t o c h r o m e c oxid a s e g e n e a n d its f l a n k i n g r e g i o n s . S e q u e n c i n g o f t h e genomic clone demonstrated the presence of Alu repetit i v e e l e m e n t s in t h e v i c i n i t y o f t h e e x o n ( m a n u s c r i p t in preparation). Southern blot hybridization of the genom i c c l o n e to r a d i o a c t i v e l y l a b e l l e d t o t a l h u m a n g e n o m i c DNA revealed the presence of multiple strongly hybridizing DNA fragments, indicating the presence of

Probe labelling and hybridization The clone c13 was labelled by nick translation using Bio-11-dUTP. After purification over a Sephadex G50-column, D N A was dissolved to a final concentration of 5 ng per ~tl hybridization mixture containing 50% (v/v) deionized formamide, 2 x SSC (1 x SSC = 150mM NaCI/15mM sodium citrate, pH 7.0), 50mM NaHPO4, 10% (w/v) dextran sulfate, final pH7.0. The in situ hybridization procedure was essentially as described by Kievits et al. (1990). The probe was allowed to pre-anneal with competitor D N A in the hybridization mixture containing sonicated herring sperm D N A (5 ~g/ml) and total human D N A (2.5 ~tg/ml) at 37~ for 2h, after a denaturation at 75~ for 5 min. Before hybridization, slides were treated under coverslip with an RNase solution (100 ~g/ml) in 2 x SSC at 37~ for l h . Then they were washed in 2 x SSC, once in PBS, and incubated in a pepsin solution (Merck, 100 ~g/ml) in 10 m M HCI at 37~ for 10 min. After washing again in PBS, a post-fixation was carried out with 4% paraformaldehyde in PBS, 50 mM MgClz, pH 7.0, at room temperature for 10min. The slides were subsequently washed in PBS, dehydrated, and air-dried chromosomal D N A was denatured by incubation of the slides in 70% v/v formamide, 2 x SSC, final pH 7.0 at 70~ for 5 min. After dehydration, 10 ~tl of the pre-annealed probe mixture was added to the slides to prevent renaturation, and hybridization took place under a 18 x 18mm eoverslip in a moist chamber at 37~ for 16h.

Detection of the biotinylated probe and banding of chromosomes Slides were washed twice in 50% (v/v) formamide, 2 x SSC, final pH 7.0,at 45~ for 10min, then twice in 2 x SSC, pH 7.0, at 45~ for 5 min, and finally once in 4 x SSC, 0.05% (v/v) Tween-20, pH 7.0, at room temperature for 5 min. All following procedures were carried out under a 24 x 50 mm coverslip. First, an incubation with 5% (w/v) non-fat dry milk in 4 x SSC, 0.05% Tween-20, at room temperature for 10 min. Then an incubation with 100 ~tl avidinFITC (5 pg/ml) in dry milk-SSC solution for 20 min. After three washes with 4 x SSC, 0.05% (v/v) Tween-20 for 5 rain each, an incubation followed with biotinylated-goat-anti-avidin in the dry milk-SSC solution for 20 rain. After a further three washes with 4 x SSC, 0.05%(v/v) Tween-20, the avidin-FITC incubation was repeated. Finally, preparations were washed twice in PBS for 5 min and mounted in 7 0 m M K-Na-phosphate buffer, pH 6.5. For R-banding a modification of the method described by Cherif et al. (1990) was used. Slides were irradiated by U V at a wave length of 365 nm using a 6 W UV-lamp from a distance of 12 cm for 20 min. After irradiation, slides were incubated in 70 m M K-Na-phosphate

Fig. 1. Mapping of biotinylated genomic sequence of human cytochrome c oxidase subunit VIb to a fluorescent R-banded human lymphocyte metaphase. A clean fluorescent spot is visible on the long arm of each chromatid of both homologues of chromosome 19

Fig. 2. Regional mapping of human cytochrome c oxidase subunit VIb to band 13.1 of chromosome 19. The diagram shows the Rband of chromosome 19. The left chromosome of each pair shows a fluorescent R-band pattern induced as described in the Materials and methods and photographed using a filterblock which is selective for the red propidium iodide-fluorescence of the chromosomes. The right chromosomes are the same chromosomes as depicted at the left, but photographed using a filterblock which is selective for the yellow green FITC-fluorescence of the probe

327 highly r e p e t i t i v e e l e m e n t s in t h e c l o n e to a t o t a l l e n g t h b f 3.4 k b ( d a t a n o t s h o w n ) . T h e r e f o r e , t h e g e n o m i c p r o b e was a l l o w e d to a n n e a l w i t h c o m p e t i t o r D N A ( h e r r i n g s p e r m a n d h u m a n ) p r i o r to in situ h y b r i d i z a t i o n to red u c e b a c k g r o u n d . T h e h y b r i d i z e d p r o b e was d e t e c t e d b y i n d i r e c t f l u o r e s c e n c e a n d r e g i o n a l m a p p i n g was a c h i e v e d by subsequent R-banding. T h e results o f t h e in situ h y b r i d i z a t i o n e x p e r i m e n t s can b e s e e n in Fig. 1. O b s e r v a t i o n o f 100 m e t a p h a s e s , to select s o m e e x a m p l e s for i l l u s t r a t i o n , s h o w e d t h a t alm o s t e v e r y m e t a p h a s e a p p e a r e d with t w o g r e e n fluoresc e n t spots, o n e on e a c h c h r o m a t i d of t h e l o n g a r m o f t h e c h o m o s o m e 19 h o m o l o g u e s . N o n e o f t h e o t h e r c h r o m o s o m e s s h o w e d c o n s i s t e n t f l u o r e s c e n t spots. S i m u l t a n e ous e x a m i n a t i o n o f f l u o r e s c e n t s p o t s a n d R - b a n d i n g p e r m i t t e d a m o r e p r e c i s e l o c a t i o n of t h e c y t o c h r o m e c o x i d a s e s u b u n i t V I b g e n e to b a n d 19q13.1. This is illust r a t e d in Fig. 2. G e n e s for s u b u n i t I V a n d V I I I o f c y t o c h r o m e c oxid a s e h a v e b e e n l o c a l i z e d o n c h r o m o s o m e 16 a n d 11, res p e c t i v e l y ( L o m a x et al. 1990; R i z z u t o et al. 1989). O u r s t u d y c l e a r l y assigns t h e s u b u n i t V I b g e n e to c h r o m o s o m e 19. T h i s result e x t e n d s t h e e v i d e n c e t h a t t h e human nuclear genes of cytochrome c oxidase are not clustered.

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