GENOMICS

10,5%-597

(19%)

Mapping of the Glycine Receptor ar2-Subunit Gene and the GABA, a34ubunit Gene on the Mouse X Chromosome JONATHAN MRC Molecular

Neurobiology

M. J. DERRY AND PENE J. BARNARD

Unit, MRC Centre, Hills Road, Cambridge

Received

October

1, 1990;

revised

January

CB2 ZQH, United Kingdom

21, 1991

has been assigned an autosomal location, the a2-subunit gene (HGM symbol: GLRAB) has been mapped to the human X chromosome (Grenningloh et aZ., 199Ob). The localization of GLRA2 to Xp21.2Xp22.1, in close proximity to the Duchenne/Becker muscular dystrophy (DMD) locus at Xp21.21, led to a predicted location for the equivalent mouse gene (proposed MGM symbol: Glra2) close to Dmd (Grenningloh et al., 199Ob), the murine homolog of the human dystrophin gene (Ryder-Cook et al., 1988). This prediction was based on the high syntenic conservation of loci on the X chromosome between mouse and man (Davisson, 1987; Amar et al, 1988; Nadeau, 1989). However, the recent report of a genetic linkage in man between the gene for the GLR a2-subunit and the hypophosphatemia (HYP, McKusick No. 30780) locus (Econs et al, 1990) may suggest a very different position for the mouse gene, not in the region of Dmd but at a telomeric position in the region of the mouse Hyp locus (Either et al, 1976). We have shown previously (Buckle et al., 1989) that the cu3-subunit of the GABA,R gene maps in close proximity to the visual pigment gene (Rsup). Using a mouse interspecific backcross between Mu..s musculus and Mus spretus described previously (Ryder-Cook et al., 1988), we have extended this analysis and have also examined the location of the GLR a2-subunit gene on the mouse X chromosome. In such crossesthe evolutionary divergence of the two genomes greatly increases the frequency of restriction fragment length polymorphisms (RFLPs), facilitating the detection and analysis of meiotic recombinations between loci. Using this strategy we have previously mapped nine reference loci and established a panel of recombinant animals (Fig. 2A) suitable for pedigree breakpoint analysis (Ryder-Cook et al., 1988). These loci include genes for cytochrome b-245 8-polypeptide (Cybb), hypoxanthine phosphoribosyltransferase (Hprt), red sensitive visual pigment (I&up), coagulation factor VIII (Cf-S), dystrophin (Dmd ), phosphoglycerate kinase-1 (Pgk-I), the mouse homolog of the human gene

We have mapped the gene for the a2-subunit of the inhibitory glycine receptor (GZro2) to the telomeric end of the mouse X chromosome by backcross analysis of a Mw musculus/Mua spretus interspecific cross. In addition, we have extended the mapping of the GABA, a3-subunit receptor gene (Gabra3). A deduced gene order of ten-Cybb-Hprt-

DXPasG-Gabra3-Rsvp-Gdx/Cf-S-Dmd-Pgk-lDXPas2-Plp-DXPasl-GIra2-tel places Gabra3 proximal to the visual pigment gene RSVPand Glra2 in the region of loci for hypophosphatemia (Hyp), steroid sulfatase (Sts), and the Ela-subunit of pyruvate dehydrogenase (Pdkal). This establishes the XF region of the mouse X chromosome as homologous with the Xp22.1-~22.3 region of the human X chromosome and indicates the presence of an evolutionary breakpoint in the region of Xp21.3. o 1991 Academic Press, Inc.

INTRODUCTION

The two amino acids glycine and y-aminobutyric acid (GABA) are the primary mediators of inhibitory neurotransmission in the central nervous system through their interaction with ligand-gated chloride channels of the neuron membrane (Barker and McBurney, 1979). These receptors share significant similarity; both have an extracellular disulfidebonded domain and four conserved transmembrane domains, suggesting a common evolutionary origin (Barnard et al., 1987). They display, however, a strikingly different distribution in the central nervous system; the glycine receptor (GLR) is found primarily in the spinal cord and brain stem, and the GABA, receptor (GABA,R) in the higher centers of the brain. The purified mammalian GLR contains two types of subunit: a 48-kDa a-subunit, which contains both agonist and antagonist binding sites (Graham et aZ., 1981, 1983; Pfeiffer et al., 1982); and a 58-kDa P-subunit, which is believed to have a structural role (Grenningloh et al., 1990a). Variants of the ligand-binding a-subunit have been cloned and while the al-subunit gene 593

Copyright 0 1991 All rights of reproduction

OSSS-7543/91 $3.ocl by Academic Press, Inc. in any form reserved.

594

DERRY

AND

BARNARD a) Glva2.1

GdX (Gdx), and the random genomic markers DXPas2 and DXPas6. This method ensures that any new probe for which an RFLP has been found can be rapidly ordered relative to known breakpoints between reference loci (Avner et al., 1987; Mock et al., 1987; Mullins et al., 1988; Barnard et al., 1990). MATERIALS

AND

METHODS

Glycine receptor probes, specific for the a2-subunit, were generated using the polymerase chain reaction (Saiki et aZ., 1988). Oligo(dT)-primed human fetal brain first-strand cDNA was amplified using oligonucleotide primer sets based on the human GLR a2-subunit cDNA sequence (Grenningloh et al., 199Ob). Two probes were generated: a 317-bp fragment, (GlyaB.l), containing mainly 5’ untranslated sequence (nts 194-510); and a 249-bp fragment. (Glya2.3), corresponding to the cytoplasmic loop (nts 1421-1669). These probes were purified from lowmelting-point agarose, oligonucleotide-labeled (Feinberg and Vogelstein, 1984), and hybridized to Hybond-N filters containing M. spretus and M. muscu1u.s DNA digested with a range of enzymes. A 7’aqI RFLP was identified for the Glya2.1 probe, with a musculus allele of 18.5 kb and a spretus allele of 11.5 kb, and an EcoRI RFLP for the Glya2.3 probe, with a musculus allele of 10.5 kb and a spretus allele of 7.0 kb. A 0.7-kb human genomic probe for the GABA, a3-subunit gene (GABA0.7Xba), containing 3’ coding and untranslated sequence, was used to reveal a TaqI RFLP, with a muscuhs allele of 10.0 kb and a spretus allele of 6.8 kb (Fig. 1). RESULTS

Using these RFLPs, we mapped the GLR a2-subunit gene in 48 backcross progeny with known recombination breakpoints from a total of more than 200 backcross progeny typed at nine reference loci (Ryder-Cook et aZ., 1988). The segregation pattern suggested a mapping position telomeric to the most distal of our reference loci, DXPas2. To investigate this further, we mapped two probes known to map telomeric to DXPas2: a probe (~27) for the proteolipid protein (Pip) gene (Milner et al., 1985), which identifies an EcoRI RFLP; and a single-copy mouse genomic probe (45) for the DXPasl locus (Amar et al., 1985), which identifies a TuqI RFLP. Seven recombinants telomeric to DXPas2 were found, indicating a most likely order of DXPas2-Plp-DXPasl -Glra%tel, and placing GZra2 distal to the random marker DXPasl (Fig. 2B). It is important to note that the selection of specified recombinant animals for backcross analysis may reduce the recombination fraction for Glra2 with any of the reference loci as a conse-

M

21.2

-

12.2

-

9.2

-

7.1

-

5.0

-

4.3

-

3.5

-

3.1

-

bl S

Glva2.3 M

c) GABA0.7Xba S

M

S

FIG. 1. RFLPs for (a) Glya2.1, (b) Glya2.3, and (c) GABA0.7Xba probes (M, Mus musculus; S, MLLS spretus). DNA was extracted by standard methods, digested with EcoRI (Glya2.3) or Tag1 (Glya2.1 and GABA0.7Xba), and transferred to Hybond-N (26). Probes were oligonucleotide-labeled (11) and hybridized to filters under conditions recommended by the manufacturer. Filters were washed in 2~ SSC/O.l% SDS (Glya2.1) or 0.5~ SSC/O.l% SDS (Glya2.3 and GABA0.7Xba) at 65°C.

quence of positive interference. We have also extended the mapping of Gabra3 using a human genomic probe (GABA 0.7Xba). Previously using a bovine GABA, cDNA probe, we localized it to the central region of the mouse X chromosome (Buckle et al., 1989) but were unable to separate Gabra3 from the Rsvp locus. Using GABA0.7Xba, we have identified a much clearer RFLP (Fig. 1) and have extended the mapping of Gabra3. We have now mapped this gene pair in 68 animals and have found a single recombinant animal that places Gabra3 proximal to Rsvp at a distance of 1.5 -+ 1.5 CM. This is consistent with the results of Hermann et al. (personal communication).

DISCUSSION

Pedigree analysis of our panel of 48 recombinant backcross progeny predicts the order cen-cybbHprt-DXPasG-Gabra3-Rsvp-Gdx/Cf-8-Dmd-Pgk-lDXPas2-Plp-DXPasl-Glra2-tel (Fig. 2). The localization of Glra2 telomeric to both Plp and DXPasl places it in the XF area of the mouse X chromosome, a region for which there have been very few gene probes previously available. Therefore, the GLRAB locus, which is close to DMD in humans, is far removed from Dmd on the mouse X chromosome, indi-

MAPPING

OF

BRAIN

RECEPTOR

595

GENES

(A) Cybb I Hprt I DXPas6 I Rsvp I Gdx/Cf-8 Dmd I Pgk- 1 I DXPas2 recomblnants total recombination freauency

0 34 23.5 *7.3

4 60 67 i3.2

175 0.6 r0.6

64 8.3 t3.0

19 127 15.0 i3.2

162 0.6 20.6

6 101 5.9 i2.4

Cybb I Hprt I DXPas6 I Gabra3 I Rsvp I Gdx/Cf-4 I Dmd I Pgk- I

I

DXPas2

I

PIP DXPas I I Glra2 331336

I

4

6

I

1-i-i

I

6

12 I8

2

2 4

2. 1 211

1

1 II

L

1 I

FIG. 2. Interspecific backcross haplotypes. (A) Haplotype distribution for nine reference loci on the mouse X chromosome. For each two-point analysis, the number of recombinants, the total number of animals typed, and the derived recombination frequency (*SE) are given (24). (B) Haplotype distribution for 48 recombinant backcross animals typed at Glra2. The total number of each haplotype is given at the base of each column. The positions of Gubra.3, Plp, and DXPasl in this subset are also given. Haplotypes are represented by filled and open boxes for Mus spretus and Mus musculus alleles, respectively.

eating that the GLRAB-DMD interval on the human X chromosome contains an evolutionary breakpoint (Fig. 3). Glra2 probes will be useful in studying an area containing a number of important disease loci, ineluding the hypophosphatemia-associated loci Hyp and Gy and the Sts locus, which is associated with

X-linked ichthyosis in man. The fine mapping of Gabra3 centromeric to Rsvp in mice raises the possibility that there may have been intrasyntenic rearrangements in this region. This speculation is based on the consensus human gene order in Xq27-q28 of HPRTDXS144-GABRA3-FW-GDX-RCP-qter (Patter-

596

DERRY 22.3a

+

AND

BARNARD

STS HYP PDHAl GLRAP DMD CYBB

A !i 6

PGKI

t-

w

RCP

HUMAN

MOUSE

FIG. 3. Mapping position of selected loci on the human and mouse X chromosomes. The probes for which mapping data are presented are underlined, and the approximate positions of Hyp, P&al, and Sts on the mouse X chromosome are shown (16). The syntenic groups in which Glra.2 and Gabra.3 map are illustrated by hatched boxes. (DXPas6 is equivalent to the human locus DXS144.)

son et al., 1987; Mandel et al., 1989), an order that may yet prove to be inaccurate. This question will be answered only when physical mapping of the Xq27-q28 region produces conclusive results.

holds (UK)

Research Council this work.

(MRC)

studentship.

The

MRC

REFERENCES 1.

ACKNOWLEDGMENTS We thank Robert Harvey for the gift of oligo(dT)-primed human fetal brain first-strand cDNA and for help with DNA sequencing, Dr. Andrew Hicks for the human GA13AA receptor a3-subunit X genomic clone, and Professor Eric Barnard for critical reading of the manuscript. The ~‘27 and 45 probes were kindly provided by Drs. Ian Milner and Phil Avner, respectively. We thank Dr. Gail Hermann for sharing her results prior to publication. J.M.J.D.

a Medical supported

2.

AMAR, L. C., ARNAUD, D., CAMBROU, J., AND GUENET, J.-L. (1985). Mapping of the mouse X chromosome using random genomic probes and an interspecific mouse cross. EMBO J. 4: 3695-3700. AMAR, L. C., DANDOLO, L., HANAUJZR, A., RYDER-COOK, A., ARNAUD, D., MANDEL, J.-L., AND AVNER, P. (1988). Conservation and reorganization of loci on the mammalian X chromosome: A molecular framework for the identification of homologous subchromosoma1 regions in man and mouse. Germmics 2: 220-230.

MAPPING

OF

BRAIN

3.

AVNER, P., AMAR, L., ARNAUD, D., HANAUJZR, A., AND CAMBROU, J. (1987). Detailed ordering of markers localizing to the Xq26-Xqter region of the human X chromosome by the use of an interspecific Mus spretus mouse cross. Proc. Natl. Acad. Sci. USA 84: 1629-1633.

4.

BARKER, J. L., AND MCBURNEY, R. N. (1979). GABA and glycine may share the same conductance channel on cultured mammalian neurones. Nature 277: 234-236. BARNARD, E. A., DAF~LISON, M. G., AND SEEBURG, P. (1987). Molecular biology of the GABA, receptor: The receptor/ channel superfamily. Trends Neurosci. 10: 502-509. BARNARD, P. J., DERRY, J. M. J., RYDER-COOK, A. S., ZANDER, N. F., AND KILIMANN, M. W. (1990). Mapping of the phosphorylase kinase alpha subunit gene on the mouse X chromosome. Cytogenet. Cell Genet. 53: 91-94. BUCKLE, V. J., FUJITA, N., RYDER-COOK, A. S., DERRY, J. M. J., BARNARD, P. J., LEBO, R. V., SCHOFIELD, P. R., SEEBURG, P. H., BATESON, A. N., DARLISON, M. G., AND BARNARD, E. A. (1989). Chromosomal localization of GABA, receptor subunit genes: Relationship to human genetic disease. Neuron 3: 647-654.

5.

6.

7.

8.

DAVISSON, M. T. (1987). X-linked genetic tween mouse and man. Genomics 1: 213-227.

9.

ECONS, M. J., PERICAK-VANCE, M. A., Bm, H., BARTLE~, R. J., SPEER, M. C., AND DWNER, M. K. (1990). The human glycine receptor: A new probe that is linked to the X-linked hypophophatemic rickets gene. Genomics 7: 439-441. EICHER, E. M., SOUTHARD, J. L., SCRIVER, C. R., AND GLORIEUX, F. H. (1976). Hypophosphatemia: Mouse model for human familial hypophosphatemic (vitamin D-resistant) rickets. Proc. Natl. Acad. Sci. USA 73: 4667-4671.

10.

11.

12.

13.

14.

homologies

RECEPTOR 15.

16.

17.

597

GRENNINGLOH, G., SCHMIEDEN, V., SCHOFIELD, P. R., SEEBURG, P. H., SIDDIQUE, T., MOHANDAS, T. K., BECKER, C.-M., AND BETZ, H. (1999b). Alpha subunit variants of the human glycine receptor: Primary structures, functional expression and chromosomal localization of the corresponding gene. EMBO J. 9: 771-776. LALLEY, P. A., DAVISSON, M. T., GRAVES, J. A. M., O’BRIEN, S. J., WOMACK, J. E., RODERICK, T. H., CREAU-GOLDBERG, N., HILLYARD, A. L., DOOLITTLE, D. P., AND ROGERS, J. A. (1989). Report of the committee on comparative mapping. Cytogenet. Cell Genet. 51: 503-532. MANDEL, J.-L., WILLARD, H. F., NUSSBAUM, R. F., ROMEO, G., PUCK, J. M., AND DAVIES, K. E. (1989). Report of the committee on the genetic constitution of the X chromosome. Cytogenet. Cell Genet. 51: 384-437.

18.

MILNER, R. J., LAI, C., NAVE, K.-A., LENOIR, D., OGATA, J., AND SUTCLIFFE, J. G. (1985). Nucleotide sequences of two mRNAs for rat brain myelin proteolipid protein. Cell 42: 931939.

19.

MOCK, B., D’HOOSTELAERE, L. A., MA~THAI, R., AND HUPPI, K. (1987). A mouse homeobox gene, Hoxl.5, and the morphological locus, Hd, map to within 1 CM on chromosome 6. Genetics 116: 607-612. MULLINS, L. J., GRANT, S. G., STEPHENSON, D. A., AND CHAPMAN, V. M. (1988). Multilocus mapping of the mouse X chromosome. Genomics 3: 187-194. NADEAU, J. H. (1989). Maps of linkage and synteny homologies between mouse and man. Trends Genet. 5: 82-86.

be-

FEINE~ERG, A. P., AND VOGELSTEIN, B. (1984). Addendum: A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Bioehem. 137: 266267. GRAHAM, D., PFEIFFER, F., AND BETZ, H. (1981). UV light-induced crosslinking of strychnine to the glycine receptor of rat spinal cord membranes. Biochem. Biophys. Res. Commun. 102: 1330-1335. GRAHAM, D., PFEIFFER, F., AND BETZ, H. (1983). Photoafhnity-labeling of the glycine receptor of rat spinal cord. Eur. J. Biochem. 131: 519-525. GRENNINGLOH, G., PIUBILLA, I., PRIOR, P., MULTHAUP, G., BEYREUTHER, K., TALEB, O., AND BE’IZ, H. (1990a). Cloning and expression of the 58 kd j3 subunit of the inhibitory glycine receptor. Neuron 4: 963-970.

GENES

20.

21. 22.

PATERSON, M., SCHWARTZ, C., BELL, M., SAUER, S., HOFKER, M., TRASK, B., VAN DEN ENGH, G., AND DAVIES, K. E. (1987). Physical mapping studies on the human X chromosome in the region Xq27-Xqter. Genomics 1: 297-306.

23.

PFEIFFER, F., GRAHAM, D., AND BETZ, H. (1982). Purification by affinity chromatography of the glycine receptor of rat spinal cord. J. Biol. Chem. 257: 9389-9393. RYDER-COOK, A. S., SICINSKI, P., THOMAS,K.,DAVIES, K. E., WORTON, R. G., BARNARD, E. A., DARLISON, M. G., AND BARNARD, P. J. (1988). Localization of the mdx mutation within the mouse dystrophin gene. EMBO J. 7: 3017-3021.

24.

25.

SAIKI, R. K., GELFAND, D. H., STOFFEL, S., SCHARF, S., HIGUCH, R., HORN, G. T., MULLIS, K. T., AND ERLICH, H. A. (1988). Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239: 487-491.

26.

SOUTHERN, E. M. (1976). Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98: 503-517.