Gene, 102 (1991) 237-244 0

1991 Elsevier

GENE

Science

Publishers

231

B.V. 0378-l 119/91/$03.50

04088

Identification of three human pseudogenes for subunit VIb of cytochrome c oxidase: a molecular record of gene evolution (Recombinant

DNA;

respiratory

chain;

nuclear

gene; mitochondrial

protein;

nucleotide

sequence

analysis;

A/u repetitive

element)

Jan-Willem Laboratory

Taanman, Cobi Scbrage, Peter Reuvekamp, Janet Bijl, Marijke Hartog, Hans de Vries and Etienne Agsteribbe of Physiological

Chemistry,

Received by H. van Ormondt: Revised: 7 February 1991 Accepted: 18 February 1991

25 August

University of Groningen,

9712 KZ

Groningen

(The Netherlands)

1990

SUMMARY

Three pseudogenes for the nuclear-encoded subunit VIb of cytochrome c oxidase (COX) were isolated by screening a human genomic library with cloned human cDNA coding for COX subunit VIb. The nucleotide sequences of the pseudogenes, designated YCOX6b-I, YCOX6b-2 and YCOX6b-3, were determined. Pseudogene YCOX6b-I bears all the hallmarks of a processed pseudogene and diverged from the parental gene after the divergence of man and cow. Alu repetitive elements were integrated into the structural sequences of the other two pseudogenes. Comparison with the human and bovine cDNA sequences encoding COX subunit VIb suggests that YCOX6b-2 and YCOX6b-3 were formed earlier in evolution than YCOX6b-I. Genomic Southern analysis indicated that a few more pseudogenes for COX subunit VIb are likely to be present in the human genome. Identical nt differences with respect to the human cDNA sequence in the pseudogenes provide some clues on the evolution of the ancestral gene coding for COX subunit VIb.

INTRODUCTION

Eukaryotic cytochrome c oxidase (COX; EC 1.9.3.1) is integrated in the mitochondrial inner membrane. The enzyme complex catalyses transfer of electrons from reduced cytochrome c to molecular oxygen coupled to proton translocation across the inner membrane in the terminal reaction of the respiratory chain (reviewed in Hatefi, 1985; Wikstrdm, 1989). In mammals, COX is composed of 13

Correspondence to: J.-W. Taanman, Laboratory of Physiological Chemistry, University of Groningen, Bloemsingel 10, 9712 KZ Groningen (The Netherlands) Tel. (31-50)632737; Abbreviations:

Fax (31-50)632606.

aa, amino acids(s),

bp, base pair(s);

plementary to RNA; COX, cytochrome COX; cpm, counts/min; kb, kilobase years;

nt, nucleotide(s);

NaCl/O.OlS

M Na,

citrate

SDS,

sodium

cDNA,

DNA com-

c oxidase; COX, gene coding for or 1000 bp; Myr, one million dodecyl

pH 7.6; Y, pseudogene

sulfate; symbol.

SSC,

0.15 M

different subunits. Like most complexes of the respiratory chain, COX is of dual genetic origin. The three largest subunits (I, II and III), which are directly responsible for the catalytic functions (reviewed in Capaldi et al., 1987; Holm et al., 1987), are encoded in the mitochondrial genome (Anderson et al., 1982) and synthesized on mitochondrial ribosomes (Hare et al., 1980). The remaining subunits (IV, Va, Vb, Via, VIb, VIc, VIIa, VIIb, VIIc and VIII, according to the nomenclature of Kadenbach et al., 1983) are of nuclear origin and synthesized in the cytosol (Hare et al., 1980) from where they are imported into mitochondria. The function(s) of the nuclear-encoded subunits is (are) obscure. Kadenbach (I 986) postulated that allosteric effeccofactors, hormones, membrane tors (e.g., metabolites, potential) change the catalytic functions by binding to specific sites at nuclear-encoded subunits. Expression of tissue-specific isoforms of COX subunits may be an additional way to modulate the oxidative energy output to suit the variable needs in different tissues. Different kinetic

238 properties of COX in liver and heart of the same animal (Merle and Kadenbach, 1982) support this possibility. The subunits Via, VIIa and VIII exist as tissue-specific isoforms (reviewed in Kadenbach et al., 1987; Yanamura et al., 1988). However, in distinction to other mammals it is likely that only one form of subunit VIII occurs in primates (Rizzuto et al., 1989). The genes coding for the subunits of mitochondrial origin have been characterized and sequenced for many species, including man (Anderson et al., 1981). To evaluate the coordination between the nuclear and mitochondrial genes involved in the biosynthesis of a functional COX complex and to study the function(s) of the nuclear-encoded subunits it is essential to characterize the nuclear genes. The analysis of the nuclear genes is complicated by the presence of processed pseudogenes. Processed pseudogenes (for review see Vanin, 1984; Weiner et al., 1986) seem to be most frequent in mammals. They completely lack introns and contain a poly(A)-tract at their 3’ end. Processed pseudogenes are flanked by short direct repeats. The characteristics suggest that processed pseudogenes have been created by reverse transcription of mature mRNA and insertion into a new chromosomal locus at a staggered break followed by repair of the target site. To be passed to subsequent generations, such events must have taken place in the germ cells or their precursors. Therefore, it is not surprising that processed pseudogenes usually are remnants of genes expressed in such cells, like genes coding for the so-called housekeeping category. Until now the following (pseudo)genes for subunits of COX have been cloned and sequenced: a bovine (Bachman et al., 1987), a chimpanzee (Lomax et al., 1990), a human (Ewart et al., 1990) and two rat (Virbasius and Scarpulla, 1990) processed pseudogenes for subunit IV, a rat (Yamada et al., 1990; Amuro et al., 1990) and the 5’ region of a bovine (Bachman et al., 1987) gene for subunit IV, two different rat processed pseudogenes for subunit VIc (Seelan and Padmanaban, 1988; Suske et al., 1988) the 5’ region of a rat gene for subunit VIc (Suske et al., 1988), and three rat processed pseudogenes for subunit VIII (Cao et al., 1989). Recently, we reported the isolation of full-length human cDNA clones coding for subunit VIb (Taanman et al., 1990). To investigate the complexity of nuclear coxlike sequences in the human genome, we have started to analyse the organization of the human COX6b gene family and described here the identification of three COX66 pseudogenes.

RESULTS

AND

DISCUSSION

(a) Isolation and sequencing for COX subunit VIb

of three human pseudogenes

To identify genomic clones containing human COX6brelated sequences, we screened a human chronic library with human full-length COX6b cDNA (Taanman et al., 1990) labeled with the random-primer extension procedure (Feinberg and Vogelstein, 1983) in presence of [c(-32P]dCTP. The library (kindly provided by Dr. G.C. Grosveld) was constructed from 15-20-kb fragments of partially Sau3A-digested human genomic DNA, cloned into 1 EMBL3 (Frischauf et al., 1983) and plated on Escherichia coli LE392. About 500000 recombinants were screened by plaque hybridization (Maniatis et al., 1982) under the stringent conditions used in Fig. 3. After rescreening of initial isolates, nine hybridizing clones were identified and plaque-purified. Seven clones, designated AGll, AG12, AGl4, AG22, /ZG31, 1G81 and AG82, were studied in detail. DNA was prepared from each isolate as described (Davis et al., 1986) and analysed by restriction mapping and Southern (1975) hybridization. The full-length fragment, a 5’ subregion (EcoRI-EcoRV fragment, nt 1-197 in Fig. 2) and a 3’ subregion (KpnI-EcoRI fragment, nt 368-450 in Fig. 2) of the cloned coxVlb cDNA were used as hybridization probes. The results indicated that the clones fall into three different groups. Of the unique clone AG81, a single 0.7-kb SalIBanzHI fragment hybridized to both the 5’- and 3’-specific probes. In view of the similarity of their restriction maps, AGl 1, i,G14 and AG82 appear to contain fragments from the same genomic region. A 1.4-kb KpnI fragment hybridized to the 5’ subregion, but not to the 3’ subregion probe in all three clones. Of these clones, /1G82 was analysed further. In AG82, a single 4.0-kb PstI fragment hybridized to both the 5’- and 3’-specific probes. Restriction mapping demonstrated that JGl2, iG22 and IG31 represented a third genomic site. In all three clones a single 6-kb SalIHind111 fragment hybridized to the 5’ subregion probe and no fragments hybridized to the 3’ subregion probe. Clone /1G22 was used for further analysis. A partial restriction map of the representatives of each group is shown in Fig. 1. Fragments that hybridized to cDNA probes in the blotting experiments are indicated. These fragments were subcloned, mapped in detail (Fig. 1) and subsequently used for sub-subcloning to facilitate sequence analysis. The sequencing strategy is shown in Fig. 1. Computer-aided nt sequence analysis showed that all clones contained YCOX66 pseudogenes. The sequence contained in AG81 is referred to as YCOX6b-I, the sequence in ilG82 is referred to as YCOX6b-2 and the sequence in AG22 is referred to as YCOX6b-3. Sequences are presented in Fig. 2.

239 (b) Structural features of !PCOX6b-2

A SK6

AQ8 1

K

H

H

Comparison of the YCOX6b-1 sequence with the cDNA sequence (Fig. 2) reveals that YCUX6b-I is colinear with

S

+cZTOXSb- 1 I

-

\

I\

SH

the cDNA except for one small insertion (nt 3 15-3 17) and two deletions of a single bp (nt 372-373 and 385-386). Thus, t,!mxVZb-I is devoid of introns. In addition, YCOX6b-1 contains a poly(A)-tract at its 3’ end

1 kb

KB

--

~ 1 kb

B A082

S

@TOXSb-9

-

KPK

PS

H

H

I

I

t /’

S I -

\

,/

lkb

\

p\ -_,-

1 kb

C

C H

S

X022

E

B

S I -

!

P I m

SP I

PP II

X 1

----

Fig. 1. Restriction maps containing COX6b-related P, &I; and

sequencing

and sequencing strategy of 1EMBL3 clones sequences. B, BarnHI; H, HindHI; K, KpnI;

of DNA strategy

sequencing.

indicate

(A) Partial

of the 20-kb genomic

the direction

restriction

map

YCOX66-1. The blackened

box denotes the 0.7-kb SalI-BumHI COX6b cDNA.

in pUCl9

to human

(Yanisch-Perron

shown below the genomic a 0.4-kb SalI-KpnI (B) Partial

below

the

fragment

COX6b cDNA

fragment

were sequenced strategy

is and

TABLE

I

as indicated.

Percent

similarities

of the 18-kb genomic

cDNA,

and the corresponding

genomic

region.

was sub-subcloned

The

0.5-kb

the coding

and

sequenced additional

3’ end of YCOX6b-2. (C) Partial restriction

probe

Human

Bovine

cDNA

cDNA

88.9 93.1

(100) 89.3

83.8

85.9

85.7

88.3

The

data of the

Bovine cDNA

of the 18.5-kb genomic

region in 1G22 which contains

YCOX66-3. The

blackened

the 6-kb SalI-Hind111

that hybridized

YCOX6b-3

strategy

was subcloned in pUCl9. A is shown below the genomic

region. There were no SphI and SmaI sites within the subcloned The 0.5-kb PsrI fragment cDNA was sub-subcloned

(blackened box) that hybridized and sequenced as indicated.

and bovine

% similarity

YTOX66-1 YCOX6b-2

to human coxVIb cDNA. This fragment detailed map of the subcloned fragment

of human

fragment

map and sequencing fragment

regions

of the three pseudogenes”

of human

as indicated. sequence

regions

box denotes

PstI-KpnI

to the 5’ subregion

was used to obtain

box denotes

between

that hybridized to human COX6b cDNA. This in pUCl9. A map of the subcloned fragment is

box) that hybridized

4.0-kb PsrI subclone

procedures fragment

YCOX6b-2. The blackened

region in 1G82 which contains

(blackened

to standard

of the subcloned

map and sequencing

the 4.0-kb PstI fragment fragment was subcloned shown

map

fragment

was subcloned

region. The 0.7-kb SalI-BumHI

sub-subcloned

restriction

This fragment

et al., 1985) according

et al., 1982). A detailed

and

region in IG8 1 which contains

that hybridized (Maniatis

PH I

1 kb

S, SalI; X, XbaI. Below each map arrows extent

X 1

(nt 697-706) and is flanked by 11-bp perfect direct repeats (5’-TTTAAATACAG). These features make YCOX66-1 a perfect example of a processed pseudogene. Like in many other processed pseudogenes (Vanin, 1984) a short poly(A)stretch of 4 nt can be found immediately upstream from the direct repeat flanking the 5’ end of YCOX6b-1 (nt 258-261). The poly(A)-tract at the 3’ end of YCOX66-1 is not aligned with the poly(A) + -tail of the cDNA presented in Fig. 2, indicating the existence of an alternative poly(A) + -site when the pseudogene arose. This alternative poly(A) + -site was also used in one of the six COX6b cDNA clones described in Taanman et al. (1990). A putative TATA box promoter element (Gannon et al., 1979; Breathnach and Chambon, 198 1) is not present in the 5’-flanking region of YCOX6b-I. However, one CCAAT box upstream promoter element (Efstratiadis et al., 1980; Dynan and Tjian, 1985) is to be found at nt 227-23 1 in the 5’-flanking region (Fig. 2). There is a 3-bp insertion at the beginning of the reading frame (nt 3 15-3 17) and there are two single bp deletions further downstream (nt 372-373 and 385-386). The resulting reading frame leads to a premature stop codon (TAA) at nt 446-448. Comparison of the coding region of the human cDNA and the corresponding region of the pseudogene reveals eight substitutions at the first position, three substitutions at the second position and seven substitutions at the third position of a codon, causing 12 aa differences and one stop codon. The overall similarity in this region is 93.1 y0 (Table I). The similarities between the 5’- and 3 ‘-noncoding regions of the

region.

to coxVIb

a The number of nt compared YCOX66-1 was 261. Any YCOX6b-3 involved tions were ignored

between human cDNA, bovine cDNA and comparison made with YCOX6-2 and

234 and 151 nt, respectively. in the calculations.

Deletions

and inser-

240 h-2 1-l e-2 e-1 'b -2

KB GCAGCCTGGG

TGACACAGTG

AGACCCTGTC

G TCGACCTGCA TCAAAARRRA RARAAAAAAA

TCTCCCTTGG CAAATCAAGA

TTTAATGTAG ATAAGACCAG

AGCAAGGGAT CCAGGCACGG

CTGAAAGCTT TGGCTCACAC

AGGGAGATTG CTGTAATCCC

GGATGGTGGA AGCACTTTGG

61 122

ti-1 6-2

GTGCATTAGT GAGGCCGAGG

CACTTTAGAC CAGGTTGATT

CTACTCATCC ACCTCAGGAG

CAGCTGGAAA GTCAGGAGTT

GGTCCAGAAG CCAGAGTAGT

ATATACCCTT CTGGCCAACT

141 182

'4-1 $-2

GACCAGTGCT TGGGGAAACC

TTGCAAAATA CTATCTCTAC

GATTTGTGAG AAAAATACAA

GGCAGCACCT AAATTAGCCA

GCATCTTTGA GGCATGATGG

AGAGCCCTGT CAGGTGCCTT

201 242

AATTGCTCTT CTCTGTATGT TAGTCCCAGC TAGTCAGGAG CTG CAGTGAGCTA

CCGATCCAAT GCCGAGGCAG TGATCATGCC

AGTGGGAATC GAGAATTGCT ACTACTCTCC

TCAGTCACTC TGAACCCACA AGCCTGAGAC

AACTACRRAA AGGCGGAGGT CCTGTCTCTG

261 302 53

AGCACCATGG M ********** AGACAGAGCA

CGG---AAGA 55 A E D ***AAG**** 321 AGACTCCTTC 362 ___**** 110

hum

GGTCAGCGGA AAAAAAGACC

21 62

TTGAGCTG

CAGGTTGAAT

CCGGGGTGCC

TTTAGGATTC

TTT-TACA TGCAGTGAGC AAAAAAGAAG

G*A****T** TGAGATCGTG ACAGCAGAGA

*TA**c**** CCGTTGCACT GTAGCCATTG

***G****** CCAGCCTGGG AGCT*AG

CATGGAGACC AAAATCAAGA MET KIK ***F;j****** G**t****** TCw AAAAAA***" *** ***k* *****A**** *** **G** ********** IQA . . .

ACTACAAGAC NY K T *******,r** ********** **,c***G**f *****c**** ..Q.

CGCCCCTTTT A P F @*******R *** Ea*p*** ****e**** it *a ****** . . .

GACAGCCGCT D S R ******Tt*X ******A*** *****G*+** ********** . . .

TCCCCAACCA F P N Q *_*'k****** ********** *?,***k**** ********** . . . .

380 422 170 79

GAACCAGACT N Q T *****_*** *******c* ********* ******** it

AGAAACTGCT R N C * *****k * *****k* * **TG*** * *****t

GGCAGAACTA W Q N Y A********* T*X**G**** *******A** **********

CCTGGACTTC LDF ******A*** ******k*** *******c** **********

CACCGCTGTC HRC ***T****** ****T****g ****A**TCA *********a

AGAAGGCAAT Q K A M ******** * ***t*** 4 * ********** ********** E...

439 482 230 139

GACCGCTAAA T A K ****A***** **T ****** *** ii****** ********** . . .

GGAGGCGATA TCTCTGTGTG CGAATGGTAC G G D I s V c E W Y *X*** **** *****+c**** *e******** **G*Al ** ***T***A** &*******,t ***** ** *A*GGCCGGG TGCAGTGGCT ***** $1*** ****c***** ******R** . . . V... . . .

CAGCGTGTGT QRV ****A***** ****C*X*h* CACGCCTGTA *G******** R..

ACCAGTCCCT YQSL ** *****i* **B ******* ATCCCAGTAC * ******* .K..

hum

CTGCCCCACA

TCCTGGGTCA

TGAGCAACGG

GCTGAAGGCA

CGTTTCC-CG

294

s-1 d-2 '3 &"

.*E**P**T* *ATT****@ TTTGGGAGGC

*:**t**si*; T**b*",," ******A*@, *,c* **fk** CAAAGCTGGT GGATTACTTA

~~~~~~

~~~~~~~~

f;:f%f$:

"6;;

hum

GGAAGATCTG G K I ********** ********** CATGATGAAA **********

hum y-1 b-2 8-3 bov

hum ,$, _1 p-2 '!, -3 bov

hum '$ -I P-2 p -3 bov

.

.

.

..I

.

.

.

._..

1..

CAGACTGGGA

.

.

.

_ 2.0 - 1.9 - 1.6 - 1,4

- 0.56

235 499 542 290 199

ST.. ATCTC-CCTT

TCCTCTGTCC

********t* ********** CCCTGTCTCT ********c*

* **_***g ********** *ii? *@a& *** ****et**** ACTAAAAATA CAAAAATTAG ** * **T*******

** ***** ***lft****** TTGGGCACGA A** *A****

****_*** * ****_** 4 * TGGCAGCTGC i***C** *

616 661 410 316

ATCATGACTT A********* ****t***** AGTTTGCCGT

409 673 719 470 359

.

5.0

175

AACTGGCTGC

.

-

115

AGGTCAGGAG TTCGAGACCA ** * ******* DR. . . .

.

.

.

-21

ACCTGGCCAA 350 ******* * 258 . . .

TCCATCCTTC

TCCC-AGGAT

352

Fig

.

hum $-1 v-2 v-3 bov

GGTGAAGGGGGACCTGG-TA +r********* l **A**A*** CTGTAATCTC ***T*f**C*

*****A*_** *******He* AGCTACTCAG ****G'

CCCAG-TGAT *** k_**** *d T e-C*** GAGGCTGAAG *T****

C-CCCACCCC k-e******* *_***X***@ CACTTGAAAC *T*'***t*

AGGATCCTAA C**k*A.* B A*C***C* TGAGAGATGG ***

hum 0-l w-2 v-3

ACCTGCTAAT CA******** G&--t+**** AAGCCGAGAT

A--AAAACTC *--******* lAT******T TGCACCACTG

ATTGGAAAAG **G**AAAAA *****_**** CAG

GTGAA&AP&i AAAAATTTAA -*e-G-

AAA . ATACAGTGGG G-G-G

i-l h-2

TCC GCCAGGTGTG

GTGG

Fig. 2. Comparison

450 731 774 493 734 788

of the nt sequences

of the human

COX6b cDNA (hum) (Taanman

f&Z) and YYXXf&-3 ($-J), and the bovine COX6b cDNA (bov) (Lightowlers by the standard

single-letter

flanking

of the YCOX6b-I

repeats

AATAACTGGA -G-GAG

code, are shown below the cDNA pseudogene

are denoted

sequences.

et al., 1990), the human pseudogenes

and Capaldi,

Identical

by arrows. The CCAAT

1989). The deduced

aa residues

in the bovine sequence

box in YCOX6b-2

WX?X6b-I

aa sequences

is overlined.

(+,-I), WX?X&Z

ofthe cDNAs,

are indicated

Ah repetitive

represented

by dots. The 1 I-bp

elements

in YCOX66-2

and YCOX66-3 are underlined. Those nt which are identical with the human cDNA sequence in the three pseudogenes or the bovine cDNA are indicated by asterisks. The nt identical in the three pseudogenes or the bovine cDNA, but not identical with the human cDNA are boxed. Dashes have been introduced in the nt sequences to improve ~ignment. For sequencing restriction fragments that contained YCOXCib-related sequences were subcloned in pUC19 and recombinant derivatives were propagated in E. cdi JM83. Recombinant plasmid DNA was isolated by the rapid boiling method (Holmes and Quigley,

1981) with an additional

phenol/chloroform

extraction.

Dideoxy

@anger

et al., 1977) double-stranded

plasmid

DNA

sequencing

was

241 human

cDNA

and

the

corresponding

regions

at the 3’ end (nt 748-773)

of the

the 5’-flanking region, we presume that YCOX66-1 is not expressed at the translation level since mutations in the coding sequence preclude the possibility that ~&oxVIb-1 could encode a functional COX subunit VIb.

in both directions

manufacturer.

A synthetic

from the IG82 submitted

subclone.

with modified

genomic

Southern

Nucleotide

blot probed

Sequence

with COX6b cDNA.

was cleaved with KpnI (K), Hind111 (H) or PsfI (P). Digested Plus filter (New England

Nuclear)

in 0.4 M NaOH/0.6

bar for 1 h. The filter was neutralized [r-“P]dCTP

created. Therefore, there is only similarity with part of the coding region of the cDNA (nt 45-198). No insertions or deletions are found in the region, however, there are eight substitutions at the first position, three substitutions at the second position and eleven substitutions at the third position of the codon in the 154 nt compared. These changes give rise to 13 aa differences. The overall similarity is 85.7% (Table I). The nt changes across the coding sequence and the integration of an Ah sequence at the 3’ end make it very unlikely that YCOX6b-3 is expressed. The presence of an Ah repetitive element at the 3’ end of the pseudogene explains why none of the clones that contained YCOX66-3 hybridized to the 3’ subregion probe. Because of the truncation at the 3’ end the possible presence of introns, a poly(A)-tract at the 3’ end and flanking direct repeats is masked. Therefore, we are unable to determine to what class this pseudogene belongs. (e) Complexity of the COX6b gene family To investigate the complexity of COX6b-related sequences in the human genome and to verify that the cloned pseudogenes had not suffered rearrangements dur-

(U.S. Biochemical

corresponding

to nt 142-159

pg sonicated

under

salmon-sperm

accession

DNA obtained

M NaCl (Chomczynski

Cleveland,

OH) according

to the protocol

of the

was used as primer to obtain additional sequence data of Mn2+ (Kristensen et al., 1990). The nt sequences have been Nos. M38259-PSIl,

from peripheral

DNA (10 wg/lane) was fractionated

in 0.4 M Tris pH 7.0/l M NaCl

denatured

Corporation,

of the cDNA,

in the presence

Databases

to a specific activity of 1 x 10” cpm/pg. The filter was hybridized

1966)/l % SDS/l00

-

YCOX6b-3 sequence almost immediately upstream from the coding region. Truncations at the 5’ end are not unusual for processed pseudogenes (Vanin, 1984). At its 3’ end YCOX6b-3 is interrupted by an Ah repetitive element which apparently integrated YCOX6b-3 after the pseudogene was

DNA derived from clone 12622 was sequenced

to the GenBank/EMBL/DDBS

Fig. 3. Human

T7 DNA polymerase

oligodeoxyribonucleotide,

with the poly(A)’

(d) Structural features of YCOX6b-3 Comparison of YCOX6b-3 with the human COX6b cDNA (Fig. 2) reveals that there is an abrupt change in the

(c) Structural features of YCOX66-2 The nt sequence analysis revealed two A/u-type repetitive elements upstream from YCOX6b-2. Ah repetitive elements (for reviews see Schmid and Jelinek, 1982; Weiner et al., 1986) are the most prominent short dispersed repeat family in primate and rodent genomes. The human Ah sequence is approx. 300 bp in length, individual elements diverge from the consensus by about 14%. Ah family members are believed to be mobile elements. Fig. 2 shows that YCOX6b-2 is interrupted by the Ah repetitive element at its 5’ end. Apparently the Ah repetitive elements have been integrated into the structural sequence of YCOX6b-2. The sequence of the 3’ end of YCOX6b-2 is colinear with the cDNA except for three small insertions (nt 600, 679 and 728-729) and three small deletions (nt 721-722, 741-742 and 745-746). Because of the interruption at the 5’ end, only the similarity with part of the coding region of the human COX6b cDNA (nt 74-305) has been determined. There are twelve substitutions at the first and second position, and 14 substitutions at the third position of the codon in the 234 nt compared, resulting in 25 aa differences. The overall similarity in this region is 83.8% (Table I). The similarity between the 3’-noncoding region of the human cDNA and the corresponding region of the pseudogene is 86.8%. The multiple genetic lesions prevent YCOX6b-2 from producing a functional protein. Because the 5’ end of the pseudogene is missing it can not be established with certainty what type of pseudogene YCOX6b-2 is. The pseudogene contains an A-rich sequence

performed

in alignment

tail of the cDNA. We assume that $coxVZb-2 is a processed pseudogene that encountered a recombination event at its 5’ end after formation in evolution, because (i) no introns are present in the region downstream from the Ah sequence, and (ii) an A-rich sequence is present at the 3’ end.

pseudogene are 82.9% and 89.8%, respectively. Although a typical transcriptional regulatory sequence is present in

and Qasba,

and air-dried.

M38260-PSI2

blood leukocytes

of one individual

on a 0.8% agarose

1984) with a Vacugene Full-length

and M38260-PS13.

Vacuum

COX6b cDNA

Blotting

to GeneScreen

Unit (LKB) at 0.05

was random-primer-labeled

with labeled COX6b cDNA in 6 x SSCj5 x Denhardt’s

DNA per ml at 65°C overnight.

(Baas et al., 1984)

gel and transferred

The filter was prehybridized

with

solution (Denhardt,

in the same solution

at 65°C

for 7 h. After hybridization the filter was washed twice in 1 x SSC at room temperature for 5 min, twice in 2 x SSCjl y0 SDS at 65°C for 30 min and twice in 0.1 x SSC at room temperature for 30 min. The filter was dried and exposed to a p-max x-ray film (Amersham) for 10 days. Later, completeness of digestion

was assured

Only hybridizing

bands

by hybridizing

the same blot with a cloned XbaI-fragment

with sizes expected

EcoRI + HindHI-digested

1 DNA. Hybridizing

the hybridizing

identical

fragment

after complete genomic

digestion

DNA fragments

in length to an internal

of human mitochondrial

were detected identical

COX6b cDNA

(results

not shown).

DNA, containing Marker

in length to those in IG81,

fragment

is indicated

IG82

by an arrow.

COX2 and 74 bp of COXl.

sizes (right; and IG22

in kb) are derived are indicated

from

by asterisks;

242 ing the cloning process, human genomic DNA was cut with the restriction enzymes KpnI, Hind111 or PstI. Digestions were analysed by Southern-blot hybridization with fulllength COX6b cDNA, under the same stringent conditions as has been used to screen the genomic library. Approximately eight hybridizing fragments were observed in each lane (Fig. 3) indicating a multigene family of moderate complexity. We isolated three pseudogenes. The 2.1-kb KpnI hybridizing fragment of IG81, the 1.4-kb KpnI and 4.0-kb PstI hybridizing fragment of AG82 and the 0.5kb PaI hybridizing fragment of AG22 correspond to similar-sized fragments in appropriately restricted (Fig. 3, asterisks). This observation

human genomic DNA ensures that none of the

clones containing the pseudogenes have been altered during construction of the genomic library. The genomic 0.15-kb KpnI fragment (Fig. 3, arrow) probably represents the internal Kpni-fragment present in coxVlb cDNA (nt 21 l-368, Fig. 2). KpnI fragments similar in size are not present in the cloned pseudogenes. Therefore, it is likely that the 0.15-kb KpnI fragment is derived from the expressed COX6b gene and thus indicates the absence of an intron in this region. Alternatively, the fragment might be derived from a fourth pseudogene. Lomax et al. (1990) and Rizzuto et al. (1989) demonstrated that COX4 and COX8, respectively, are present as single-copy genes. Like for COX subunit IV and COX subunit VIII there are no tissue-specific forms for COX subunit VIb (Taanman et al., 1990). If we assume that COX6b is also present as a single-copy gene, the hybridization pattern in Fig. 3 allows the presence of a few additional pseudogenes. (f) Evolutionary considerations The existence of cox pseudogenes appears to be a widespread phenomenon in mammals. In addition to the ten processed pseudogenes isolated by others (Bachman et al., 1987; Seeland and Padmanaban, 1988; Suske et al., 1988; Cao et al., 1989; Lomax et al., 1990; Ewart et al., 1990; Virbasius and Scarpulla, 1990) we characterized three pseudogenes. Comparison of the nt changes between pseudogenes and cDNA can be used to estimate the divergence time. However, the small number of nt that can be compared for the pseudogenes we isolated leads to large variability in the estimated time of divergence. Using the method developed by Saccone et al. (1990) it was calculated that YCOX6b-I diverged from the parental gene 45 + 32 Myr ago. The large standard deviation illustrates the limited usefulness of the calculation for short sequences. Recently, Lightowlers and Capaldi (1989) isolated a cDNA clone specifying bovine COX subunit VIb. Since the similarity between the coding region of bovine cDNA and the corresponding region of YCOX6b-1 is 89.3-93.1% with respect to the human cDNA (Table I), YCOX6b-I is clearly

more closely related to the human gene than to the bovine gene. Therefore, we assume that YCOX66-I arose after the human line diverged from the bovine line, which has been estimated some 80 Myr ago. There is no doubt that YCOX66-2 and YCOX6b-3 were formed at an earlier time than YCOX6b-1. Table I shows that the similarities between YCOX6b-2 and YCOX6b-3, and bovine cDNA are in fact higher than the similarities between the pseudogenes and human cDNA. These data suggest that YCOX6b-2 and YCOX6b-3 arose prior to the divergence of man and cow. Comparisons of the nt sequences of human and bovine cDNA, and the three pseudogenes show that there are 22 positions in the coding region where the pseudogenes or the bovine cDNA have identical nt differences with respect to human cDNA (boxed nt in Fig. 2). We assume that these differences reflect mutations in the human gene. Thirteen of the 22 differences concern third codon positions, eleven differences do not affect the aa sequence. These data indicate that the coding region of the human COX6b gene is under selective constraint, otherwise the number of differences in each codon position would be equal (Kimura, 1983). The identical nt differences provide an impression of the sequence of the ancestral gene and its evolution. Some of the identical nt differences are present in all three pseudogenes, e.g., the T 125+ C transition or the A12’ + G transition in human cDNA (Fig. 2). Other identical nt differences are only present in the older two pseudogenes, e.g., the G19* -+ A transition or the A195 --f G transition in human cDNA (Fig. 2). Identical nt differences in all three pseudogenes suggest relatively recent changes in the human gene, whereas identical nt differences only present in the older two pseudogenes suggest relatively old changes in the human gene.

NOTE ADDED

Since the article was submitted, we learned that two human pseudogenes for subunit VIb of cytochrome c oxidase were characterized by others (Carrero-Valenzuela). One of the two pseudogene clones (IHCOX61) corresponds to our YCOX6b-2, except that nt 527 of $coxVIb-2 is deleted in AHCOX61. The second pseudogene clone characterized by Carrero-Valenzuela et al. (1991) is different from the pseudogenes identified in this paper.

ACKNOWLEDGEMENTS

We thank Dr. G.C. Grosveld (Erasmus University, Rotterdam) for providing the human genomic library, Dr. Graziano Pesole (Universita degli Studi, Bari) for computer analysis, Siert Knollema for initial screening of the library,

243 Harry

Bakker

for preliminary

genomic

Southern

experi-

restriction

ments, Marijke Holtrop for useful discussions, Bert Tebbes for photography and Karin Klappe for providing peripheral

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Identification of three human pseudogenes for subunit VIb of cytochrome c oxidase: a molecular record of gene evolution.

Three pseudogenes for the nuclear-encoded subunit VIb of cytochrome c oxidase (COX) were isolated by screening a human genomic library with cloned hum...
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