GENOMICS

11,302-308

(1991)

Chromosomal Localization of a Cytochrome b, Gene to Human Chromosome 18 and a Cytochrome b, Pseudogene to the X Chromosome E. A. SHEPHARD,*,’ S. PovEv,t N. K. SPURR,$ AND I. R. PHILLIPS§ *Department of Biochemistry and Molecular Biology, University College London, Gower Street, London WClE 6BT, United Kingdom; tMRC Human Biochemical Genetics Unit and the Galton Laboratory, University College London, Wolfson House, 4 Stephenson Way, London NW7 2HE, United Kingdom; *Human Genetic Resources, Imperial Cancer Research Fund C/are Hall Laboratories, South Mimms, Hefts. EN6 3LD, United Kingdom; and §Department of Biochemistry, Queen Mary and Westfield College, University of London, Mile End Road, London El 4NS, United Kingdom Received

November

26, 1990;

Isolation of Human

METHODS

Cytochrome b, cDNA Clones

DNA Sequencing The cDNA inserts of several of the plasmids isolated from the human cDNA library were subcloned into M13mp18 and 19 and sequenced by the dideoxy chain termination method (Sanger et aZ., 1980) using either the Klenow fragment of Escherichiu coli DNA polymerase or modified T7 polymerase (Sequenase, United States Biochemicals, Cleveland, OH).

Polymerase Chain Reaction Genomic DNA was isolated from human leukocytes, a Faza rat hepatoma cell line, and human-rodent somatic cell hybrids as previously described (Ed-

be addressed.

o&M-7543/91 $3.00 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.

AND

A human liver cDNA library (Woods et al., 1982) was plated out and screened as described previously (Phillips et al., 1985). A 32P-labeled rat cytochrome b, cDNA clone (Rigby et rd., 1989) was used as the hybridization probe at a concentration of 5 rig/ml. After hybridization the final wash of the filters was for 1 h at 5O’C in 1 M NaCl/O.l% SDS.

Cytochrome b,, which is present in virtually all cells, is located mainly in the membranes of the endoplasmic reticulum, where it functions as the intermediate electron carrier in the NADH-dependent stearyl-CoA desaturase system (Strittmatter et al., 1974). It can also serve as the second electron donor to cytochrome P450 monooxygenases (Imai and Sato, 1977) involved in the metabolism of endogenous compounds such as steroids and prostaglandins and of foreign compounds including drugs, chemical carcinogens, and environmental pollutants (Ortiz de Montellano, 1986). In the cytosol of mature erythrocytes the protein is present in a truncated soluble form that, together with NADH-dependent cytochrome b5 reductase (EC 1.6.2.2), is responsible for the reduction of methemoglobin (Hultquist and Passon, 1971), and

should

13, 1991

MATERIALS

INTRODUCTION

correspondence

May

there is at least one reported case of an inherited methemoglobinemia due to a deficiency of cytochrome b, (Hegesh et al., 1986). In this paper we report the use of a technique based on the restriction endonuclease digestion of products amplified by a polymerase chain reaction (PCR) to determine the chromosomal localization of the human cytochrome b, gene(s) and one of its processed pseudogenes.

We have isolated cDNA clones that code for human cytochrome b, . Owing to the high degree of evolutionary conservation of cytochrome b5 sequences and the existence of human and rodent cytochrome b, processed pseudogenes, we were unable to map unambiguously the chromosomal localization of the human gene(s) by Southern blot hybridization of DNA from human-rodent somatic cell hybrids. An alternative approach, baaed on restriction enzyme digestion of PCR-amplified DNA, enabled us to map the human cytochrome bB gene(s) to chromosome 18 and one of its processed pseudogenes to the X chromosome. We propose the designations CYB5 and CYBbPl for the gene and pseuo issi Academic PWSS, IBIC. dogene loci, respectively.

1 To whom

revised

302

MAPPING

OF

HUMAN

CYTOCHROME

wards et al., 1985). DNA was isolated from mouse liver as described by Blin and Stafford (1976). DNA samples (2 pg) were amplified for 30 cycles essentially as described by Abbott et al. (1988) using either Thermus aquuticus (Taq) DNA polymerase (2.5 units/ml) (Cetus) or Thermw thermophitus (Tet) DNApolymerase (5 units/ml) (All-Union Research Institute of Influenza, Leningrad, USSR). Each cycle consisted of 1 min each at 90, 46, and 70°C. Reactions were performed either manually using three water baths set at the appropriate temperatures or automatically in a Hybaid thermal reactor. The priming oligonucleotides were synthesized on a Cruachem DNA synthesizer and their sequence is shown in Fig. 1.

b5 GENE

2:

AND

303

PSEUDOGENE

PstI HaeIII CCTGCCATCElGCAGTGGCCGTCGCClTGATGTATC G A ATCAAGGCA AA T T A A CC TG A TC

1. 2. 3. 4.

GCCTATACATGGCAGAGGAC@&CACCTCCTCAGAAGTCAGCGCAGGAAGAGCCTGC C--AG C T AT G A A A C T A C A TA CTGTCTG C CAGA AA

:: 3. 4.

T~TGGA~ACGGG.~G~~GAAG~A~G~TAACTA~~~AA~TGACAG.~,AMXT A A CA A T C A A A CCCA G G G T TC C

A C

FIG. 1. DNA sequence of the product amplified from the human cytochrome h, gene. The termination codon is boxed. The sequences of the human cytochrome b, pseudogene 1 (line 2) and pseudogene 2 (line 3) and the rat cytochrome b5 gene (line 4) are given only at positions where they differ from that in line 1. The sequences of primers 1 and 2 are underlined.

Analysis of PCR Products The amplified products were extracted once with an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1, v/v/v) and the DNA was precipitated with ethanol at -20°C. Pellets were washed once with 80% ethanol, dried, and resuspended in 10 mMTris, 1 n&f EDTA (pH 8.0). Restriction enzyme digestion of the PCR products was carried out according to the suppliers’ recommendations. DNA samples were analyzed by electrophoresis through a 2% agarose gel followed by ethidium bromide staining. In some cases, DNA fragments were excised from the gel and the DNA was recovered by centrifugation through siliconized glass wool. The recovered fragments were subjected to further digestion by restriction enzymes and the products analyzed by agarose gel electrophoresis as described above.

Hybrids The somatic cell hybrids used have all been previously described (Table 1) but many have been subcloned since having been originally described. RESULTS

Isolation of Human

Cytochrome

b5 cDNA Clones

Screening of 5 X lo4 colonies of a human liver cDNA library with a rat cytochrome b, cDNA identified several positive colonies. After two additional rounds of screening at lower colony density five clones were selected for further analysis. The nucleotide sequences of all five of the cDNAs proved to be identical in their overlapping regions (data not shown). The longest cDNA isolated (700 bp) encoded all but the first three N-terminal amino acids of cytochrome b, , followed by a termination codon, 287 bp of 3’ noncoding sequence, and a poly(A) tail. The sequence was identical to that reported by Yoo and Steggles (1988). Analysis of five additional cy-

tochrome b, cDNAs by restriction enzyme mapping revealed no differences and we found no evidence in this library for cDNAs derived from partially processed mRNAs as reported by Yoo and Steggles (1989).

Chromosomal Localization Cytochrome b5 Gene

of a Human

Our initial approach to determining the chromosomal localization of the human cytochrome. b, gene(s) was to analyze, by Southern blot hybridization, DNA isolated from a panel of independent human-rodent somatic cell hybrids. A complex pattern of DNA fragments hybridized to the human cytochrome b, cDNA (data not shown) and we were unable to assign unambiguously the chromosomal localization of the corresponding gene(s). We therefore adopted an alternative strategy based on restriction enzyme analysis of products of PCR amplification. This method allows us to discriminate between human and rodent cytochrome b, genes and known human (Yoo and Steggles, 1989) and rat (J. Rigby, E. A. Shephard, and I. R. Phillips, unpublished) cytochrome b, processed pseudogenes. Based on the human cytochrome b, cDNA sequence two PCR primers were selected that would amplify a 170bp product from the human cytochrome b, gene (Fig. 1; Fig. 2, track a). The PCR product extends from the first nucleotide of the codon for amino acid residue 109 and includes 89 3’ noncoding nucleotides. From a knowledge of the structure of a rat cytochrome b, gene (Rigby et al., 1989) the amplified DNA lies within the last exon of the gene. The sequence of primer 1 is identical to the corresponding regions of the rat cytochrome b, gene (J. Rigby, E. A. Shephard, and I. R. Phillips, unpublished) and the human cytochrome b, pseudogene 2 (Yoo and Steggles, 1989), and has one mismatch with the human cytochrome b, pseudogene

304

SHEPHARD

ET

AL.

the hybrid FIR5Cl (whose only human chromosome is 14) was not (Fig. 2, track l), confirming the assignment of the human cytochrome b, gene(s) to chromosome 18. Chromosomal Localization of a Human Cytochrome b5 Pseudogene FIG. 2. Analysis of DNA products amplified by the polymerase chain reaction using primers 1 and 2 (Fig. 1). DNA samples were from man (o), mouse (p), Faza cells (q), and the somatic cell hybrids FIR5 (b), HORL411B6N4 (c), lA9498 (d), 2WIR70 (e), 3W4clIO (f), FST9/16 (g), FSTS/lO (h), MOG2E5 (i), SIF4A24El (j), SIF15P5 (k), FIR5Cl (1). HORLSX (m), FIRBRBG (n), FGlO (8). The PCR products were incubated with PstI and electrophoresed through a 2% agarose gel. Track (a) shows the PCR product amplified from human DNA but not incubated with PatI. Track (r) shows molecular weight standards. Numbers refer to sizes of the PCR product and digested fragments in bp.

1 sequence (Yoo and Steggles, 1989) (Fig. 1). Primer 2 has two mismatches with the corresponding regions of the rat cytochrome b, gene and human cytochrome b, pseudogene 1, and one mismatch with human pseudogene 2 (Fig. 1). Thus in addition to amplifying a 170-bp region of the human cytochrome b, gene the primers would be expected, under the conditions used, to amplify the corresponding regions of rat (Fig. 2, track q) and mouse (Fig. 2, track p) cytochrome b, genes and of both the known human cytochrome b, pseudogenes. As there is little homology between primer 2 and the rat processed pseudogene, the latter would not be amplified. Only the product amplified from the human cytochrome b, gene will contain a PstI site (Fig. 1). The presence of this site in the PCR products can therefore be used to map specifically the chromosomal location of the human cytochrome b, gene. Digestion with P&I of the PCR product amplified from human DNA yields fragments of 170,134, and 36 bp in length (Fig. 2, track 0). The 36-bp fragment was frequently not seen clearly on the gels. Neither the rat nor mouse amplified products were digested by this enzyme (Fig. 2, tracks q and p). Therefore the presence of a fragment of 134 bp after PstI digestion of products amplified from human-rodent somatic cell hybrids was taken to indicate the presence of the human cytochrome b, gene. An analysis of 26 human-rodent somatic cell hybrids revealed that the presence of a Pat1 site in the amplified DNA products cosegregated with human chromosome 18 (Fig. 2, Table 1). Eleven of the hybrids were positive for the PstI site and all of these contained human chromosome 18. The DNA product amplified from one of the hybrids (FIR5RB6) (whose only human chromosomes are 14 and 18) was digested by Pat1 (Fig. 2, track n), whereas that amplified from

The 170-bp fragment present after PstI digestion of the PCR product amplified from human genomic DNA represents cytochrome b,-like sequences that do not contain a PstI site (Fig. 2, track 0). To map the chromosomal location of these sequences we have utilized an Hue111 site that is present in all the known human cytochrome b, gene sequences but is absent from rat and mouse cytochrome b, gene sequences (Fig. 1). HaeIII digestion of the PCR product amplified from human genomic DNA yields only two fragments, 130 and 40 bp in length, only the larger of these being clearly visible on the gel (Fig. 3A, track b). This confirms that all the human cytochrome b, DNA sequences that have been amplified do indeed contain the HaeIII site. The rat PCR product is not digested by this enzyme (Fig. 3A, track d), whereas Hue111 digestion of the mouse PCR product yields a small fragment that is easily distinguished from the human DNA fragments (Fig. 3A, track c). In the absence of human chromosome 18 (which carries the cytochrome b, gene, as described above) the presence of Hoc111 site in the amplified gene product can be taken to indicate the presence of one or more pseudogenes. Amplified product from all 26 hybrids described in Table 1 was digested with HaeIII and scored for the presence of the 130-bp fragment, which would indicate the presence of a HaeIII site. The results from 17 of the hybrids are shown in Table 2. As expected all hybrids containing human chromosome 18 were pbsitive but because this method does not allow the detection of pseudogenes in the presence of the coding gene they are not informative and all except two have been omitted from the table. The two exceptions, FIR6RB6 and TWINlSF9, containing chromosome 18 but not the X chromosome, were selected for further study. PCR products from these hybrids were first digested by PstI to cleave sequences amplified from the cytochrome b, gene itself (located on human chromosome 18). Amplified DNA that remained uncut (170 bp) was resolved by agarose gel electrophoresis from the products of digestion (134 and 36 bp) and excised from the gel. Incubation of this material with Hue111 showed that it did not contain a Hue111 site, thus indicating that none of the known human cytochrome b, pseudogenes is present on chromosome 18. Inspection of Table 2 shows complete correlation of the presence of a cytochrome b, pseudogene with the

MAPPING

OF

HUMAN

CYTOCHROME

b,

TABLE Segregation

Hybrid

Ref.

FSTS/lO FST9/16 MOG2E5 MOG2C2 FGlO FGlOE8EP2-7 DUR4.3 TWINlSF9 HORL411B6N4 FIR5 FIR5RB6 HORLSX lA9498 2WIR70 3W4cllO SIF4A24El SIF15P5 FIR5Cl cl21 762-8A PNTSl MCP6 640-63a12 Jlc14 289 C4A/G

(16) (16) (24)

Cytochrome Concordant ++ --

b5/chromosome

(24)

(16) (1‘3 (23) (27) (28)

(25) (25) (28) (2:) (18)

(6) (6) (25) (4)

(8) (29) (9) (14) (15) (17) (7)

of Human

PCR product digested with PstI + + f + + + + + + + + -

Discordant +-+

Cytochrome

GENE

AND

1

b, in 26 Human-Rodent Human

1

2

3

4

5

6

7

8

9

--++-+-+ +

10

Somatic

Cell Hybrids

chromosome 11

12

13

14

15

16

+

+

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

-

-

17

18

19

+

+

-

20

21

22

X

5 12

77 15 8

/+-++++--+-+-++

-++-+---+-+--+--+-+-++ + + +

+ -

305

PSEUDOGENE

+

+

+

+

+

-

-

/+-I++/-++-/++/

-

-_

,

-

-

+++-+--+---+-+

Q--------------

-

+++++++++--

-

f -

-

-

+ +

/-+/+----+--+--+-+ /-+,-----+--+--,-+

-

-

+

-

-

-

-

-

/+++

++++-+-++--+/+--++-+++--+-------+-+----+---++ -------------+---+-----

-

-

-_-

-

-

/-/------+---+----I -

-

/-------+

-----+-----f--------+-+ -

-

-

_ ---+------------+---+-+ -

-

-

-

+---+-+---------,

-

_

-

+

+---++--+---++----+--+

-------------+-------+------+------------------------+----------------+------------------

-

-

-

-

/----------------,

-

-

-

-

-

-

-

-

-

-

-

-

-

-

-

--

5 12

2 11

6 14

4 8 241032

5

-

+------------

3 14

5 12

3 12

5 13

4 13

7 12

6 13

2 15

5

5

8

6 2

6 2

4 3

4 1

9 0

------------+----------------+---------------

3 15

2 14

7 15

5 14

4 14

8 0

9 1

4 0

6 1

6 1

Note. +, Human chromosome present; -, human Q, long arm of chromosome present. o Gift of D. Cox, University of California.

chromosome

not detected;

X chromosome, and at least three examples of discordance with all other chromosomes. Two hybrids provide some evidence for regional assignment. The presence of a cytochrome b, pseudogene in the hybrid MCPG, whose only human component is der(6)t(X;6)(q13;p21), localizes the gene to Xq13qter. The positive result in hybrid 2WIR70, which contains an unidentified fragment of the X chromosome that is positive for PGK and negative for HGPRT and GGPD (Van Heyningen et aZ., 1975), does not conflict with this conclusion. DISCUSSION We have isolated cDNA clones that code for human cytochrome b,. In contrast to a report by Yoo and

/, equivocal

results,

part

411 13 15 7 2

of chromosome

14 15 14

010 0 0 present,

5 1

5 3

42 05

or not tested.

Steggles (1989), we found no evidence for cDNAs derived from partially processed mRNAs. This is probably due to differences in the methods of RNA extraction and/or cDNA synthesis used in the construction of the different libraries that were screened. Several factors may contribute to our inability to map the chromosomal localization of the human cytochrome b, gene(s) by Southern blot hybridization of DNA isolated from human-rodent somatic cell hybrids: (i) the gene contains at least five exons (some of which are very small) separated by relatively large introns (Rigby et al., 1989), (ii) the existence of at least two closely related pseudogenes in man (Yoo and Steggles, 1989) and one in rat (J. Rigby, E. A. Shephard, and I. R. Phillips, unpublished), and (iii) a possible EcoRI polymorphism associated with the hu-

306

ET

SHEPHARD

abcde

fgh

localization of genes coding for p68 nuclear antigen (Iggo et al., 1989) and complement component C9 (Abbott et al., 1989). As an alternative approach to mapping the cytochrome b, gene(s) we used a method based on the amplification of genomic DNA by a PCR. Previous reports of chromosomal mapping by PCR have exploited species-specific differences in the length of an amplified intron (Iggo et al., 1989) or used oligonucleotide primers whose sequence would allow amplification of the corresponding gene from human but not from rodent DNA (Abbott et al, 1989; Dionne et al., 1990). Neither of these methods is suitable for the cytochrome b, gene due to the large size of its introns, the high degree of evolutionary conservation of the exon sequences, and the lack of information on the intron sequences of the human gene. The approach that we have taken involves the use of primers that will amplify all known human and rodent cytochrome b, genes and pseudogenes, and relies on the presence or absence of specific restriction endonuclease sites in the various amplified fragments to distinguish those

abcdef

130.

LO-

FIG. 3. DNA products amplified by the polymerase chain reaction using primers 1 and 2 (Fig. 1). Amplified products were incubated with Hue111 and electrophoresed through a 2% agarose gel. (A) DNA samples were from man (b), mouse (c), Faza cells (d), and the somatic cell hybrids HORL411B6N4 (f), lA9498 (g), 2WIR70 (II). Track (e) shows molecular weight standards. (B) DNA samples were from the somatic cell hybrids MCP6 (a), C4a/G (b), 289 (c), HORLSX (d), cl21 (e). Track (a) shows the PCR product amplified from human DNA but not incubated with HacIII. Track (f) shows molecular weight standards. Numbers refer to sizes of the digested fragments in bp.

man cytochrome b5 gene (data not shown). Similar difficulties have been encountered in the use of Southern blot hybridization to map the chromosomal TABLE Segregation

Hybrid 3W4cllO lA9498 2WIR70 SIF15P5 HORLSX SIF4A24El MCP6 FIR5RB6* TWINlSFS* cl21 PNTSl 640-63q12 Jlc14 289 FIR5Cl 762-8A CQA/G Cytochrome Concordant ++ -Discordant +-+

Ref.

W-3) (2;)

(6) (28) (6) (9) (25) (27) (4) (29) (14) (15) (17)

of a Cytochrome

PCR product digested with Hue111

1

2

+

------+--+

+

_____

+ + + + + _

(25) (8)

_

(7)

-

3

b, Pseudogene

4

5

6

7

8

AL.

2 in 17 Human-Rodent

9

10

Somatic

Cell Hybrids

Human

chromosome

11

12

13

14

15

16

17

18

19

20

21

22

X

+

+

-

+

-

-

+

-

-

-

+

-

+-

-

-

T

-

Y

+-----+--------++++

+--v--+---------T-++--++--++--++

----

+--+-

-,---,-------+-+------------+---+++_

,

_

_

_

_

++++-+-++-

-

--++~--------------____

---+

+

-

-

-

-

-

-

-

-

/

+---+-----+ -

-

+

+

-

+++--

+

-

-

-

-

-

-

-

-

-

-

-

+----------

-

-

-

-

-

-

_

+

9

8

0130 10 9

9

9

50 10 9

5

7

---------------Q---------------

_

_

-

_

-

_

-

-

-

_

-

-

-

-

-

-

---+

-

-

-

+

+-------------

-

-

____

-

-----_------

b, pseudogene/chromosome 01010230021212 9 9 9 9 7676744775 11111112111113

9

9

9

8

8

9

9

9

8

7

5

5

6

5

1020 10 10 5 7 0012

764 0111

7

07 01

Note. +, Human chromosome present; -, human chromosome not detected; /, equivocal results, part of chromosome present, or not tested. Q, long arm of chromosome present. T, relevant translocation, see text. a Gift of D. Cox, University of California. * The PCR product was first digested with P&I, and the uncut 170-bp fragment was excised from an agarose gel then digested with Has111 as described in the text. The negative scoring for HaeIII digestion refers to the PstI-resistant 170-bp fragment.

MAPPING

OF HUMAN

CYTOCHROME

of human origin from those of rodent origin and the human gene from its pseudogenes. Thus we have exploited a PatI site unique to sequences amplified from the human cytochrome bS gene to map this gene to chromosome 18. The sequences of the two human cytochrome b, processed pseudogenes identified to date (Yoo and Steggles, 1989) suggest that both would be amplified by the PCR primers used and that the amplified products would be digested by the restriction endonuclease HueIII. We have used this approach to map at least one of these pseudogenes to the X chromosome. However, we cannot exclude the possibility that additional human cytochrome b, pseudogenes exist, the sequences of which either cannot be amplified by the primers used or, if amplified, lack an Hoe111 site. There also might be additional pseudogenes on chromosome l&16, or 19, since hybrids containing these chromosomes and lacking both X and 18 were not tested. According to the published sequences (Yoo and Steggles, 1989) it should be possible to distinguish between the two human cytochrome b, processed pseudogenes by RsaI digestion of their PCR-amplified DNA products; that from pseudogene 2 would be digested, whereas that from pseudogene 1 would not (Fig. 1). However, we obtained no digestion by RsaI of DNA amplified from 12 different individuals (data not shown). As the sequence of pseudogene 2 has no mismatches with primer 1 and only one mismatch with primer 2 it is unlikely that it did not amplify, especially as the rat cytochrome b, gene (which has more mismatches with the primers) was amplified. Thus either the individuals analyzed did not possess this pseudogene or there is an error in the reported sequence. ACKNOWLEDGEMENTS We thank Dr. Peter Swarm for synthesizing the oligonucleotides used in this study and the USSR Ministry of Health All-Union Research Institute of Influenza for Tet polymerase. This work was supported by grants from the Medical Research Council. REFERENCES ABBOT,

C. M., MCMAHON, C. J., WHITEHOUSE, D. B., AND S. (1988). Prenatal diagnosis of alpha-1-antitrypsin deficiency using polymerase chain reaction. Lancet 1: 763POVEY, 764.

ABBOT, C., WEST, L., POVEY, S., JEREMIAH, S., MURAD, Z., DISC~O, R., AND Fcr, G. (1989). The gene for human complement component C9 mapped to chromosome 9 by polymerase chain reaction. Genomics 4: 606-609. BLIN, N., AND STAFFORD, D. W. (1976). Isolation of high molecular weight DNA. Nucleic Acids Res. 9: 2982-2998. CROCE, C. M., AND KOPROWSKI, H. (1974). Somatic cell hybrids between mouse peritoneal macrophages and SV40

b, GENE

5.

6.

7.

8.

9.

307

AND PSEUDOGENE

transformed human cells. 1. Positive control of the transformed phenotype by the human chromosome 7 carrying the SV40 genome. J. Erp. Med. 140: 1221-1229. DIONNE, C. A., KAPLAN, R., SEUANFZ, H., O’BRIEN, S. J., AND JAYE, M. (1990). Chromosome assignment by PCR techniques: Assignment of the oncogene FG-5 to human chromosome 4. Biatechniques 8: 190-194. EDWARDS, Y. H., PARKAR, M., POVEY, S., WEST, L. F., PARFUNGTON,J. M., AND SOLOMON, E. (1985). Human myosin heavy chain genes assigned to chromosome 17 using a human cDNA clone as probe. Ann. Hum. Genet. 49: 101-109. EDWARDS, Y. H., BARLOW, J. H., KONIALIS, C. P., POVEY, S., AND BUTTERWORTH, P. H. W. (1986). Assignment of the gene determining human carbonic anhydrase, CAI, to chromosome 8. Ann. Hum. Genet. 50: 123-129. FISHER, J. H., KAo, F. T., JONES, C., WHITE, R. T., BENSON, B. J., AND MASON, R. J. (1987). The coding sequence for the 32000-Dalton primary surfactant-associated protein A is located on chromosome 10 and identifies two separate restriction fragment length polymorphisms. Am. J. Hum. Genet. 40: 503-511. GOODFELLOW, CHAMBERS,

P.

N.,

BANTING,

G.,

TR~WSDALE,

J.,

S., AND SOLOMON, E. (1982). Introduction of a human X-6 translocation chromosome into a mouse teratocarcinoma: Investigation of control of HLA-A, B, C expression. Proc. Natl. Acad. Sci. USA 79: 1190-1194. 10. HEGESH, E., HEGESH, J., AND KAFFORY, A. (1986). Congenital methemoglobinemia with a deficiency of cytochrome bs. N. Engl. J. Med. 314: 757-761. 11. HULTQUIST, D. E., AND PASSON, P. G. (1971). Catalysis of methaemoglobin reduction by erythrocyte cytochrome b6 and cytochrome b, reductase. Nature (New Biol.) 229: 252-254. 12. ICGO, R., GOUGH, A., Xu, W., LANE, D. P., AND SPURR, N. K. (1989). Chromosome mapping of the human gene encoding the 68-kDa nuclear antigen (p68) by using the polymerase chain reaction. Proc. Natl. Acad. Sci. USA 86: 6211-6214. 13. 1~1, Y., AND SATO, R. (1977). The roles of cytochrome b, in a reconstituted N-demethylase system containing cytochrome P-450. B&hem. Biaphys. Res. Commun. 76: 420-426. 14. JONES, C., KAO, F. T., AND TAYLOR, R. T. (1980). Chromosomal assignment of the gene for folylpolyglutamate synthetase to human chromosome 9. Cytogenet. Cell Genet. 28: 181194. KAo, F. T., JONES, C., AND PUCK, T. T. (1976). Genetics of 15. somatic mammalian cells: Genetic, immunologic, and biochemical analysis with Chinese hamster cell hybrids containing selected human chromosomes. Prac. Natl. Acad. Sci. USA 73: 193-197. KIELTY, C. M., POVEY, S. K., AND HOPKINSON, D. A. (1982). 16. Regulation of expression of liver specific enzymes. III. Further analysis of a series of rat hepatoma and human somatic cell hybrids. Ann. Hum. Genet. 46: 307-327. 17. MOLGAARQ H. V., SPURR, N. K., AND GREAVES, M. F. (1989). The haemopoetic stem cell antigen, CD34, is encoded by a gene located on chromosome 1. Leukaemia 3: 773-776. 18. NABHOLZ, M., MIGGIANO, V. K., AND BODMER, W. F. (1969). Genetic analysis with human-mouse somatic cell hybrids. 19.

Nature ORTIZ

223: 358-363. DE MONTELLANO,

P-450: Structure, New York. 20.

PHILLIPS,

P. R. (Ed.) (1986). “Cytochrome Mechanism, and Biochemistry.” Plenum,

I. R., SHEPHARD,

E. A., ASHWORTH,

A., AND RABIN,

B. R. (1985). Isolation and sequence of a human cytochrome P-450 cDNA clone. Proc. Natl. Acad. Sci. USA 82: 983-987.

308 21.

22.

23.

24.

25.

26.

21.

SHEPHARD RIGBY, J. S., BULL, P. C., ASHWORTH, A., SHEPHARD, E. A., SANTISTEBAN, I., AND PHILLIPS, I. R. (1989). Isolation and characterization of genes coding for cytochrome b5 and cytochrome b, reductase. Biochem. Sot. Trans. 17: 194-195. SANGER, F., COULSON, A. R., BARRELL, B. G., SMITH, A. J. H., AND ROE, B. A. (1980). Cloning in single-stranded bacteriophage as an aid to rapid DNA sequencing. J. Mol. Btil. 143: 161-178. SOLOMON, E., BOBROW, M., GOODFELLOW, W. F., SWALLOW, D. M., POVEY, S., AND Human gene mapping using an X/autosome Somatic Cell Genet. 2: 125-140.

P. N., BODMJZR, NOEL, B. (1976). translocation.

SOLOMON, E., SWALLOW, D., BURGESS, S., AND EVANS, L. (1979). Assignment of the human ol-glucosidase gene (olGLu) to chromosome 17 using somatic cell hybrids. Ann. Hum. Genet. 42: 273-281. SOLOMON, E., HIORNS, L., CRYSTAL, R., AND SYKES, the human (~2(1) collagen lar hybridization. Cytogenet. STRITTIWATTER, P., SPATZ, SETLOW, B., AND REDLINE, ties of rat liver microsomal Proc. N&l. Acad. Sci. USA SWALLOW, D. M., POVEY, HARRIS, H., PYM, B., AND

DALGLEISH, R., TOLSTOSHEV, P., B. (1983). Regional localization of gene on chromosome 7 by molecuCell Genet. 36: 64-66. L., CORCORAN, D., ROGERS, M., R. (1974). Purification andproperstearyl coenzyme A desaturase. 71: 4565-4569. S., PARKAR, M., ANDREWS, P. W., GOODFELMW, P. (1986) Mapping

ET

AL. the gene coding for the human liver/bone/kidney alkaline phosphatase to chromosome 1. Ann. 229-235.

Hum.

isozyme Genet.

of 50:

28.

VAN HEYNINGEN, V., BOBROW, M., BODMJZR, W. F., GARDINER, S. E., POKEY, S., AND HOPKINSON, D. A. (1975). Chromosome assignment of some human enzyme loci: Mitochondrial malate dehydrogenase to 7, mannosephosphate isomerase and pyruvate kinase to 15 and probably esterase D to 13. Ann. Hum. Genet. 38: 295-303.

29.

VARESCO, L., THOMAS, H. J., WILLIAMS, S., FENNELL, S. J., HOCKEY, A., SEARLE, S., BODMJZR, W. F., FFUSCHAUF, A.-M., AND SOLOMON, E. (1989). Clones from a deletion encompassing the adenomatous polyposis coli gene (APC): Human Gene Mapping 10. Cytogenet. Cell Genet. 61: 1098. WOODS, D. E., MARKHAM, A. F., RICKER, A. T., GowBERGER, G., AND COLTEN, H. R. (1982). Isolation of cDNA clones for the human complement protein factor B, a class III major histocompatibility complex gene product. Proc. Natl. Acad. Sci. USA 79: 5661-5665.

30.

31.

32.

Yoo, M., ANJJ STEGGLES, A. tide sequence of human liver Biophys. Res. Commun. 156: Yoo, M., AND STEGGLES, A. of partially processed mRNA liver cytochrome br,. B&hem. 18-24.

W. (1986). The complete nucleocytochrome be mRNA. Biochem. 576-580. W. (1989). The characterization and two pseudogenes for human Biophys. Res. Commun. 163:

Chromosomal localization of a cytochrome b5 gene to human chromosome 18 and a cytochrome b5 pseudogene to the X chromosome.

We have isolated cDNA clones that code for human cytochrome b5. Owing to the high degree of evolutionary conservation of cytochrome b5 sequences and t...
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