GENOMICS 8,568-570 (1990)

SHORT COMMUNICATION

The a! Subunit of Cytochrome b_,,, Mapped to Chromosome 16

HANAN N. Bu-GHANIM,* COLIN M. CASIMIR,*,’ SUE POVEY,~ AND ANTHONY W. SEGAL*

*Department of Medicine, Faculty of Clinical Science, University College London, London, WCIE 6JJ, United Kingdom; and tMRC Human Biochemical Genetics Unit, the Galton Laboratory, University College London,

Wolfson House, 4 Stephenson Way, London NW1 2HE, United Kingdom

Received December 7, 1989; revised June 26, 1990

The chromosomal location of the (Y subunit (33~kDa pro- tein) of human cytochrome b-,,, was analyzed by Southern blot hybridization using DNA isolated from a panel of 12 independent human-rodent somatic cell hybrids. The re- sults indicate that this protein is encoded at a single locus

on chromosome 16. o lee0 Academic press, tne.

Cytochrome b-,, is the terminal component of the NAPDH oxidase electron transport chain of phago- cytic cells that is activated when these cells engulf bacteria and is important for their microbicidal func- tion. This oxidase system does not function in chronic granulomatous disease (CGD), which results in fre- quent infections because of diminished defense against a wide variety of microorganisms (Holmes et aZ., 1967). Cytochrome b_,,, is absent from the cells of patients with the more common X-linked form of CGD and is present in normal levels in those with an autosomal recessive pattern of inheritance (Segal et al., 1983). Cytochrome bzd5 is a heterodimer, com- posed of a 76- to 92-kDa heavily glycosylated p sub- unit (Parkos et al., 1987; Segal, 1987) whose gene is situated on the X chromosome (Wolff et al., 1980) and a 23-kDa a heme binding subunit (Yamaguchi et aZ., 1989; Nugent et al., 1989). RNA coding for the a sub- unit appears to be present in many cell types in addi- tion to those of the myeloid series; however, the activ- ity of this protein is detectable only in phagocytic cells that express the larger subunit (Parkos et al., 1988).

In this study we have determined the chromosomal localization of the gene coding for the 23-kDa (Y sub- unit. Three types of rodent-human somatic cell hy-

1 To whom correspondence should be addressed.

brids were used; rat-human, mouse-human, and hamster-human somatic cell hybrids. The hybrids used in this work are listed in Table 1.

DNA samples were digested according to the sup- plier’s recommended conditions (Boehringer-Mann- heim) with restriction endonucleases EcoRI, HindHI, or BanHI (3 units/pg DNA over 18 h) and fraction- ated by electrophoresis through a 1% agarose gel. DNA fragments were transferred (Southern, 1975) to nylon membranes (Hybond-N, Amersham Interna- tional) and hybridized with 32P-labeled cDNA from the (Y subunit. Membranes were washed to a final stringency of 0.1X SSC (1X SSC is 0.15 MNaCl, 0.015 M sodium citrate, pH 7.0)/0.1% SDS at 55°C and au- toradiographed at -70°C using high-performance au- toradiography film (Hyperfilm-MP, Amersham) with intensifying screens for 3 to 7 days.

The probe used was a full-length cDNA of the (Y subunit of cytochrome b-245, 680 bp long (Parkos et aZ., 1988). It was labeled by nick-translation (BRL Kit) to a specific radioactivity of 4-9 x lo8 cpm/pg with [(u-32P]dCTP.

To determine the best restriction digest that iden- tifies the human (Y subunit from the rodent (Y subunit fragments, human, mouse, rat, and hamster genomic DNAs were analyzed by Southern blot hybridization after digestion with EcoRI, NindIII, and BamHI re- striction endonucleases (data not shown). The BamHI restriction digest was chosen because upon hybridization with the (Y subunit cDNA probe, it gave two fragments in the human DNA of 16.6 and 1.3 kbp, which were well distinguished from all other frag- ments in mouse, rat, and hamster BamHI restriction digests.

In DNA from 5 of the 13 independent hybrids ana- lyzed [MOG2E5, MOG2C2 (Solomon et al., 1979), FST7 (Kielty et al., 1982), TWINlSD12 (Swallow et

osss-7543/90 $3.00 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

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TABLE 1

The Segregation of the Gene for the a Subunit of Cytochrome b-,,, in Relation to Chromosomal Content in 13 Independent Human-Rodent Somatic Cell Hybrids

Human chromosome

Ref. 23K 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 X

MOG2E5 (13) + + - + + + + + + / + - + + + + + + + - / + / +

MOG2C2 (13) + + - + + + - + + + + + - - + + + + + + / + / + FST7 (4) + - - - - - - + - - + + + - + - + + + + + - + + TWINlSD12 (15) + + - + + - + - + - - - + - + - + + + - + + + -

2860H7 (5) + - -__-_____-_- --+ -_- - - - -

TWINlSF6 (15) - - + + + - + - + + - - + / + - - + + - + + + -

FST9/2 (4) - - - + - - + / + - + - + - + + - - + - - - + + SIF15P5 (1) - - + - - - + +-- f---+ + - - - - + - - +

SIF4A24El (10) - - - - + - - - _ - - - - - - - _ + - - - + - +

HORL411B6 (16) _ + _ + - - - - - - - + - + - + - - + - - - + +

HORP9.5 (16) - - _ - - - - - _ - + + + - + _ _ - _ _ - _ + +

3W4CL5 (6) - ------+ --+ + f-+ + - + - - - +-+

64063a12 (3) - --- - - - --+ - - --- -_- - - - - - -

++ 30332233132314254422323 7 6 5 6 85566454634855 8 6 5 4 2

+- 25223322323241301131212 -+ 12 3 2 0 3 2 2 2 4 3 41 5 4 0 3 3 0 2 3 4 6

Discordance (%) 23 54 38 31 23 46 33 31 42 46 46 46 42 46 54 0 31 31 23 27 38 45 62

Note. f, human chromosome present; -, human chromosome not present; /, not tested or equivocal results.

al., 1986), and 2860H7 (Mulley and Cullen, 1986); see Table 11, two hybridizing fragments identical in size to the fragments in human DNA were obtained. No hybridization of the (Y subunit probe was observed in

12 3

FIG. 1. Southern blot hybridization of BumHI restriction di- gests of human DNA (track l), and a positive (track 2) and a nega- tive (track 3) mouse-human somatic cell hybrid. The size of the fragments is in kbp.

DNA from the other hybrids, other than cross-hybrid- izing rodent fragments. Examples of the positive and negative hybrids are shown in Fig. 1.

The segregation of human chromosomes in the hy- brids is shown in Table 1. (Some of the hybrids used have been regrown and recharacterized since they were first described.) The results clearly indicate that the presence of the (Y subunit of cytochrome b-,, is correlated with the presence of human chromosome 16. Each of the other chromosomes displays a discor- dance of between 23 and 64%.

In accordance with the previous assignment for the fl subunit of the cytochrome, we suggest the nomen- clature CYBA for the (Y subunit locus.

ACKNOWLEDGMENTS

We thank the Wellcome Trust for their support, Mary Dinauer and Stewart Orkin for providing the a subunit cDNA clone, and Nigel Spurr for providing some of the hybrids,

REFERENCES

1. EDWARDS, Y. H., PARKER, M., PO~EX, S., WEST, L. F., PARRINGTON, 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.

2. HOLMES, B., PAGE, A. R., AND GOOD, R. A. (1967). Studies of the metabolic activity of leukocytes from patients with a ge- netic abnormality of phagocyte function. J. Clin. Invest. 48: 1422-1432.

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3.

4.

5.

6.

7.

8.

9.

10.

11.

JONES, C., AND KAO, F. T. (1983). Regional mapping of the folylpolyglutamate synthetase gene (FPGS) to Seen-q34. Cy- togenet. Cell Genet. 37: 499.

KIELTY, C. M., POVEY, S., AND HOPKINSON, D. A. (1982). Regu- lation 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.

MULLEY, J. C., AND CULLEN, D. E. (1986). New regional local- izations for HAGH and PGP on human chromosome 16. Hum. Genet. 74: 423-424.

12

13.

NABHOLZ, M., MIGGIANO, V., AND BODMER, W. F. (1969). Ge- netic analysis with human-mouse somatic cell hybrids. Nu- ture (London) 223: 358-363.

14.

NUGENT, J. H. A., GRATZER, W., AND SEGAL,, A. W. (1989). Identification of the haem binding subunit of cytochrome

b-245. Biochem. J., in press.

15.

PARKOS, C. A., ALLEN, R. A., COCHRANE, C. G., AND JESATIS, A. J. (1987). Purified cytochrome b from human granulocyte plasma membrane is comprised of two polypeptides with rela- tive molecular weights of 91,000 and 22,000. J. Clin. Znuest. 80: 732-742.

16.

PARKOS, C. A., DINALJER, M. C., WALKER, L. E., ALLEN, R. A., JESATIS, A. J., AND ORKIN, S. H. (1988). Primary structure and unique expression of the 22-kilodalton light chain of hu- man neutrophil cytochrome b. Proc. Natl. Acad. Sci. USA 86: 3319-3323.

SANTISTEBAN, I., POVEY, S., WEST, L. F., PARRINGTON, J. M., AND HOPKINSON, D. A. (1985). Chromosome assignment, bio- chemical and immunological studies on a human aldehyde dehydrogenase, ALDH3. Ann. Hum. Genet. 49: 87-100.

SEGAL, A. W. (1987). Absence of both cytochrome b-245 sub-

17

18.

units from neutrophils in X-linked chronic granulomatous disease. Nature (London) 326: 88-91.

SEGAL, A. W., CROSS, A. R., GARCIA, R. C., BORREGAARD, N., VALERIUS, N., SOOTHHILL, J. F., AND JONES, 0. T. G. (1983). Absence of cytochrome b-245 in chronic granulomatous dis- ease: A multicenter European evaluation of its incedence and relevance. N. En&. J. Med. 308: 245-251.

SOLOMON, E., SWALLOW, D., BURGESS, S., AND EVANS, L. (1979). Assignment of the human acid n-glucosidase gene ((uGLU) to chromosome 17 using somatic cell hybrids. Ann. Hum. Genet. 42: 273-281.

SOUTHERN, E. M. (1975). Detection of specific sequences among DNA fragments seoerated by gel electrophoresis. J. Mol. Biol. 98, 503-517.

SWALLOW, D. M., POVEY, S., PARKAR, M., ANDREWS, P. W., HARRIS, H., PYM, B., AND GOODFELLOW, P. (1986). Mapping of the gene coding for the human liver/bone/kidney isozyme of alkaline phosphatase to chromosome 1. Ann. Hum. Genet. 50: 229-235.

VAN HEYNINGEN, V., BOBROW, M., BODMER, W. F., GAR- DINER, S. E., POVEY, S., AND HOPKINSON, D.A. (1975). Chro- mosome assignment of some human enzyme loci: Mitochon- drial malate dehydrogenase to 7, mannose-phosphate-isom- erase and pyruvate kinase to 15 and probably, esterase D to 13. Ann. Hum. Genet. 38: 295-303.

WOLFF, G., MULLER, C. R., AND JOBKE, A. (1980). Linkage of

genes for chronic granulomatous disease and Xg. Hum. Genet. 54: 269.

YAMAGUCHI, T., HAYAKAWA, T., KANEDA, M., KAKINLJMA, K., AND YOSHIKAWA, A. (1989). Purification and some properties of the small subunit of cytochrome b,,, from human neutro- phils. J. Biol. Chem. 264: 112-118.