GENOMICS

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(19%)

The GABA, Receptor @3 Subunit Gene: Characterization of a Human cDNA from Chromosome 15qll q13 and Mapping to a Region of Conserved Synteny on Mouse Chromosome 7 J. WAGSTAFF,* J. R. CiiAiLLET,tr$

AND M. LALANDE*+’

*Division of Genetics, Children’s Hospital, 300 Longwood Avenue, -IDepartment of Genetics, Harvard Medical and *Howard Hughes Medical institute, Boston, Massachusetts 02175 Received

May8,

INTRODUCTION

GABA (y-aminobutyric acid), the principal inhibitory neurotransmitter in the vertebrate brain, exerts its inhibitory effects by binding to GABA, receptors, which are ligand-gated chloride channels. GABA, receptors appear to be both functionally and structurally heterogeneous. Recently, a large number of GABA, receptor subunit genes have been isolated and characterized ((~1-6; 01-3; yl, 2; 6; ~1) (reviewed

correspondence

should

1991

in Olsen and Tobin, 1990; seealso Cutting et al., 1991) and expression of combinations of these genes in transfected cells has led to the appearance of functional GABAA receptors (see for example Schofield et al., 1987). These receptor subunit genes show distinct, but in some cases overlapping, patterns of gene expression in the brain. However, the subunit composition and stoichiometry of GABAA receptors in vivo has not yet been determined. We have recently localized the GABA, receptor 03 subunit gene (GABRB3) to human chromosome 15qllq13 (Wagstaff et aZ., 1991), a region that is frequently deleted in two distinct genetic syndromes, Angelman syndrome (AS) and Prader-Willi syndrome (PWS) (Ledbetter et aZ., 1981; Kaplan et cd., 1987; Magenis et aZ., 1987). Deletions of the maternal chromosome 15 in this region are associated with Angelman syndrome, whereas deletions of the paternal chromosome are found in patients with Prader-Willi syndrome (Butler and Palmer, 1983; Knoll et al., 1989). These observations have led to the suggestion that one or more genes in this region are differentially expressed on maternally versus paternally inherited chromosomes, a phenomenon referred to as genetic imprinting (Knoll et al., 1989; Nicholls et cd., 1989). In all patients examined in our study with either AS or PWS due to interstitial deletions, the GABRBS gene was deleted. However, we found that this gene was intact in a PWS patient with an unbalanced 9;15 translocation, suggesting that deletion of the GABRB3 gene may not be required for the PWS phenotype (Wagstaff et cd., 1991). In this report, we describe the isolation and characterization of a human GABRBS cDNA clone and show that the Gabrb-3 gene is located on mouse chromosome 7, a chromosome that shows imprinting effects. Gabrb-3 lies in a region of conserved synteny that contains other loci from human 15qllq13.

A cDNA encoding the human GABA, receptor 83 subunit has been isolated from a brain cDNA library and its nucleotide sequence has been determined. This gene, GABRBS, has recently been mapped to human chromosome 15qllq13, the region deleted in Angelman and PraderWilli syndromes. The association of distinct phenotypes with maternal versus paternal deletions of this region suggests that one or more genes in this region show parentalorigin-dependentexpression (geneticimprinting).Comparison of the inferred human /33 subunit amino acid sequence with 83 subunit sequences from rat, cow, and chicken shows a very high degree of evolutionary conservation. We have used this cDNA to map the mouse 83 subunit gene, Gabrb-3, in recombinant inbred strains. The gene is located on mouse chromosome 7, very closely linked to Xmv-33 between Tam-l and Mtv-I, where two other genes from human 15qllql3 have also been mapped. Thi’s provides further evidence for a region of conserved synteny between human chromosome 15qllql3 and mouse chromosome 7. Proximal and distal regions of mouse chromosome 7 show genetic imprinting effects; however, the region of homology with human chromosome 15qllq13 has not yet been associated with these effects. o Is81 Academic Crete, IIN.

’ To whom

School,

he addressed. 1071

Copyright 0 1991 All rights of reproduction

o&B-7543/91 $3.00 by Academic Press, Inc. in any form reserved.

1072

WAGSTAFF, 1 61

CHAILLET,

AND

LALANDE

CGTCGCGACGGCGGCGGGGCGCCCCCTCCCCCGTGCCGGGGCGCGGCG~G~TG~GG~ GCCTTGCGGGAGGAAGGCTTTTCGGCATCTTCTCGGCCCCGGTGCTGGTG~TGTGGTGT LAGGPLFGIFSAPVLVAVvC

121

GCTGCGCCCAGAGTGTGAACGATCCCGGGAACATGTCCTT CAQSVNDPGNMSFVKETVDK

181

AGCTGTTGAAAGGCTACGTCGCCTAAGACCCC~CTTC~GGGTCCCCCGGTCTGCG LLKGYDIRLRBDFGGPPVCv

241

TGGGGATGAACATCGACATCGCCAGCATCGACATGGTTTCCGAAGTCAACATGGATTATA GMNIDIASIDMVSEVNMDYT

301

CCTTAACCATGTATTTTCAACAATATTGGAGAGATAAAAGGCTCGCCTATTCTGGGATCC LTMYFQQYWRDKRLAYSGIP

361

CTCTCAACCTCACGCTTGACAATCGAGTGGCTGAGTGGCT~C~GCTATGGGTGCCCGA~CATATT LNLTLDNRVADQLWVPDTYF

421

TCTTAAATGACUAAAGTCATTTGTGCATGGAGTGACAGTGAAAAACCGCATGATCCGTC LNDKKSFVHGVTVKNRMIRL

481

TTCACCCTGATGGGACAGTGCTGTATGGGCTCAGAATCAC HPDGTVLYGLRITTTAACMM

541

TGGACCTCAGGAGATACCCCCTGGACGAGCAGAACTGCAC DLRRYPLDEQNCTLE

601

GCTACACCACGGATGACATTGAGTTTTACTGGCGAGGCGG YTTDDIEFYWRQGDKAVTGV

661

TGGAAAGGATTGAGCTCCCGCAGTTCTCCATCGTGGAGCAG ERIELPQFSIVEHRLVSRNV

721

TTGTCTTCGCCACAGGTGCCTATCCTCGACTGTCACTGAGCTTTCGGTT~~G~~ VFATGAYPRLSLSFRLKRNI

781

TTGGATACTTCATTCTTCAGACTTATATGCCCTCTATACT~T~C~TTCTGTCGTGGG GYFILQTYMPSILITILSWV

841

TGTCCTTCTGGATCAATTATGATGCATCTGCTGCTAGAGTTG SFWINYDASAARVALGITTV

901

TGCTGACAATGACAACCATCAACACCCACCCACCTTCGG~~C~TGCC~TCCCCTATG LTMTTINTHLRETLPKIPYV

961

TCAAAGCCATTGACATGTACCTTATGGGCTGCTTCGTCTTTGTGTTCCT~CCCTTCTGG KAIDMYLMGCFVFVFLALLE

1021

AGTATGCCTTTGTCAACTACATTTTCTTTGGAAGAGGCCCCTC~GGCAG~G~GCTTG YAFVNYIFFGRGPQRQKKLA

1081

CAGAAAAGACAGCCAAGGCAAAGAATGACCGTTCAAAGAGCGAAAGCAACCGGGTGGATG EKTAKAKNDRSKSESNRVDA

1141

CT~AT~G~TATTCTGTTGACATCGCTGGAAGTTCACAAT ILLTSLEVHNEMNEVSG

1201

GCGGCATTGGCGATACCAGG~TTCAGCAATATCCTTTGA~CTCAG~TCCAGTACA GIGDTRNSAISFDNSGIQYR

1261

GGAAACAGAGCATGCCTCGAGAAGGGGCATTCCTCCTCCCGC K Q S M P REGHGRFLGDRSLPH

1321

ACAAGAAGACCCATCTACGGAGGAGGTCTTCACAGCTCAAAATTAAAATACCTGATCTAA KKTHLRRRSSQLKIKIPDLT

1381

CCGATGTGAATGCCATAGACAGATGGTCCAGGATGGTCCAG~TCGTGTTTC~~T~CT~TT~CT~TT~ DVNAIDRWSRIVFP

1441

TCAACTTAGTTTACTGGCTGTACTATGTTAACTGAGTGAC NLVYWLYYVN

1501

CTTCATTTAACACTGAGTGAAATATTATTACTCTGCCTGTC~GTTTTTATACCTGTA~CAC

1561

ACAGACACACAAGCAGACACACACATATATATACATACGC~TTGTATATATATGTG~CTT

1621

CTCAGCATATATAT

FIG. 1. Nucleotide sequence of human GABA* indicated. The 5’ untranslated region extends from positions 1467to 1634.

1634 receptor positions

83 subunit cDNAs and inferred amino acid sequence. Nucleotide 1 to 53, and the sequenced portion of the 3' untranslated region

positions are extends from

GABRBB

cDNA

MAPS

TO

MOUSE

CHROMOSOME

1073

7

MWGLAGGRLFGIFSAPVLVAVVCCAQSVNDPGNMSFVKETVDKLLKGYDIRLRPDF F F F RL FG I

Hum31 Rat31 Bov31 Chi31

ADQLWVPDTYFLNDKKSFVHGVTVKNRMIRLHPDGTVLYGLRITTTAACMMDLRRY

Hum143 Rat143 Bov143 Chi143

PLDEQNCTLEIESYGYTTDDIEFYWRGGDKAVTGVERIELPQFSIVEHRLVSRNW

Hum199 Rat199 Bov199 Chi199

N

Y

K

FATGAYPRLSLSFRLKRNIGYFILOTYMPSILITILSWVSLGI

Hum255 Rat255 Bov255 Chi255

M

-RETLPKIPYVpYIFFGRGP

QRQKKLAEKTAKAKNDRSKSESNRVDAHGNILLTSLEVHNEM--NEVSGGIGDTRN I APMD N GD S N I RF GS T

VAS

A SV A V TTSVT

K

Hum311 Rat311 Box7311 Chi311

A

Hum365 Rat365 Bov365 Chi367

***** SAISFDNSGIQYRKQSMPREGHGRFLGDRSLPH-KKTHLRRRSSQLKIKIPDLTDV K YM I R HMV T SH SL RSS TGS S RG

Hum420 Rat420 Bov419 Chi423

NAID-VFPFTFB

Hum448 Rat448 Bov447 Chi451

M

FIG. 2. Comparison of deduced positions are indicated, and negative The four transmembrane regions between transmembrane regions 3 (33); and chicken sequence is from

MATERIALS

I

amino acid sequences from human, rat, bovine, and chicken GABA, receptor p3 subunits. Amino acid numbers refer to signal peptide residues. Only residues that differ from the human sequence are shown. are underlined, and the presumed regulatory site for protein kinase A in the large intracellular region and 4 is indicated by asterisks (*). Human sequence is from Fig. 1; rat and bovine sequences are from Ref. Ref. (2).

AND

METHODS

PCR Isolation of a /33-Specific Probe from Human DNA Primer sequences from the rat GABA, receptor /?3 subunit cDNA sequence (Lolait et al., 1989; Ymer et

al., 1989) were 5’-GTTGGTGACACCAGGAATTCAGC-3’ and B-GTACAGCCAGTAAACTAAGTTG3’. These primers amplify a 25%bp fragment, corresponding to a portion of @3exon 9, from human genomic DNA (Wagstaff et al., 1991). Amplification was performed in a Perkin-Elmer-Cetus cycler, with 30 cycles of 1 min at 94”C, 1 min at 6O”C, and 90 s at

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CHAILLET,

AND

LALANDE

Southern Blot Analysis of DNA from Inbred Strain-s and Somatic Cell Hybrids

kb

Mouse

DNA from inbred mouse strains was cleaved with restriction enzymes, separated on agarose gels, and transferred to nylon filters (GeneScreen, DuPont). Hybridization solutions contained 40% formamide, and filters were washed at 55°C in 2X SSC/O.l% SDS. Probes included the 258-bp PCR product described above and a 0.7-kb EcoRI-XhoI fragment from the 5’ end of the JP5 cDNA insert, and were labeled by random-primer DNA synthesis (Feinberg and Vogelstein, 1983).

2.62.3-

RESULTS

Isolation and Characterization cDNAs FIG. 3. Autoradiograph of Southern blot of Sty1 digests of genomic DNA from C57BL/6J and DBA/PJ mouse strains. The blot was hybridized with a 0.7-kb EcoRI-XhoI probe from the 5’ end of the JP5 cDNA and shows a variation in the size of the largest band between these two strains. The approximate sizes of the polymorphic bands are 2.3 kb in C57BL/6J (Lane 1) and 2.6 kb in DBA/BJ (Lane 2).

Approximately 2 X 10’ phage from a human brain cDNA library were hybridized with a PCR-generated probe specific for the GABA, receptor /33 subunit

TABLE BXD

72°C. The PCR product was purified in a 0.8% lowmelting-temperature agarose gel, and labeled by random-primer DNA synthesis (Feinberg and Vogelstein, 1983). The labeled product detected a single band on Southern blots of human genomic DNA cleaved with the restriction enzyme Hind111 (data not shown). Isolation

of GABRB3

cDNA

Clones

A human fetal brain cDNA library constructed in vector XGTlO was screened with the 258-bp probe prepared by PCR amplification of human genomic DNA with primers from the rat GABRBS gene (see above). Hybridization, purification of positive phage, and preparation of phage DNA were performed according to standard procedures (Sambrook et al., 1989). Two phage, JP5 and JPlO, containing the largest inserts (3.0 kb) were selected for further analysis. DNA

Sequencing

Fragments from the JP5 and JPlO inserts were subcloned into pBS(SKII)+ (Stratagene) or into Ml3 vectors. DNA sequencing was performed with a Sequenase 2.0 kit (USB) according to manufacturer’s recommendations. In some cases, solutions containing 7-deaza-dGTP (USB) were used in these reactions.

of Human GABRB3

Strain 1 2 5 6 8 9 11 12 13 14 15 16 18 19 20 21 22 23 24 25 27 28 29 30 31 32

1

Strains

Mtv-I

Xmv-33

Gabrb-3

Tam-l

B D B B B B B D B D D B B B B D D B B D B B B B D D

B D B B B B B D B D D B B B B B D B B D B B B B D D

B D B B B B B D B D D B B B B B D B B D B B B B D D

B D D D B B B D B D B B B B B B D B B D B D B B D D

X

X X

X

X

Note. B is the allele from C57BL/6J and D is the allele from Mtv-1 and Tam-l types are from Ref. (30). Xmv-33 types

DBA/BJ. are from loci.

Ref. (9). X indicates

a recombination

event

between

two

GABRB3 CHROnOSOME7

MA

\L

cDNA

MAPS

TO

PP

T9H

T50H

MOUSE

CHROMOSOME

gepe. There were 19 hybridizing phage clones, and DNA was prepared from 6 of these. The largest inserts (3.0 kb) were found in phage ,JP5 and JPlO. DNA sequence analysis of subclones from JP5 showed an open reading frame encoding a protein highly homologous to @3 receptor proteins from rat and bovine sources (Ymer et aZ., 1989). However, this homology extended only from amino acid positions 3 to 448; these positions correspond exactly to those encoded by exons 2 through 9 of the GABA, receptor 81 subunit gene, whose genomic organization has recently been determined (Kirkness et aZ., 1991). Immediately 5’ to this open reading frame was a consensus splice acceptor site (CTCGCAG) , suggesting that this cDNA was synthesized from an incompletely spliced mRNA. Sequence analysis of the second cDNA, JPlO, showed a 5’untranslated region of 53 basepairs and an open reading frame encoding a sequence highly homologous to the inferred N-termini of rat and bovine

1075

p3 receptor subunits (Ymer et al., 1989). A composite cDNA sequence from JP5 and JPlO is shown in Fig. 1. The amino acid sequence inferred from these human j33 cDNAs shows a very high degree of conservation when compared to reported sequences from rat (97% identity), cow (97%), and chicken (92%) (Fig. 2; Lolait et al., 1989; Ymer et al., 1989; Bateson et al., 1990). As the comparison of amino acid sequences in Fig. 2 shows, the sequence differences between species are clustered near the carboxyl terminus of the receptor protein, in what is predicted to be a large intracellular domain. The number of amino acid substitutions in the presumptive extracellular and membrane spanning domains is very small. Two previously described human genomic clones containing 03 exons (Wagstaff et aZ., 1991) showed exon-intron boundaries to be precisely conserved between the p3 and /31 genes for exons 4 and 6. The corresponding sequence of this cDNA is identical to the exon sequences from these genomic clones, confirming that the genomic clones were from the /33 gene.

Mapping

FIG. 4. Map of mouse chromosome 7, showing the localization of Gubrb-3 between Tam-l and Mtv-I. Map positions of markers Tam-l, p, Mtu-I, and c are from Ref. (7). Positions of the breakpoints in translocation strains T(7;15)9H and T(7;18)50H are from Ref. (27). D?Hmsl was mapped to the position shown by ChaiIlet et al. (5) and D7Nicl was mapped by Nicholls (21). Imprinting maps of chromosome 7 are from Ref. (28).

7

of the Gabrb-3 Gene in Mouse

The P3-specific PCR-generated probe described above was hybridized to a panel of DNAs from six mouse-hamster hybrid cell lines digested with Hind111 in order to localize the Gabrb-3 gene in the mouse. In addition to a 6-kb hamster band, three hybrids showed hybridization to a mouse-specific band

at 16 kb. These hybrids shared only mouse chromosome 7 (data not shown). To localize the gene more precisely on chromosome 7, this probe was used to search for RFLVs (restriction fragment length variants) between inbre’d mouse strains C57BL/6J and DBAJSJ. Although approximately 50 restriction enzymes were tested; no RFLV was found using this probe. A 017-kb EcoRI-XhoI subclone from the 5’ end of the JP5 cDNA insert was, however, found to detect a Sty1 polymorphism between these two inbred strains (Fig. 3). This probe was used to map Gubrb-3 with recombinant inbred strains (Taylor, 1969). Among 26 BXD recombinant inbred strains, there were no recombinants with Xmv-33 on chromosome 7 (Table l), and there was close linkage to Mtv-1 (1.0 * 1.0 CM) (Fig. 4). This is the same location previously determined for the D7Hmsl locus, defined by a mouse probe isolated by homology to the human cDNA clone DN34, which lies within the AS and PWS critical regions (Chaillet et al., 1991). This is also the same map position determined for locus D7Nicl, defined by human cDNA probe DNlO (Nicholls, 1989). This cDNA was iso-

1076

WAGSTAFF,

CHAILLET,

lated by homology to genomic clone pIRlO-1, which lies near, but not within, the AS and PWS critical regions (Tantravahi et al., 1989; Wagstaff et al., 1991; J. H. M. Knoll and M. Lalande, unpublished results). DISCUSSION Previous studies localizing GABA, receptor subunit genes in the human (Buckle et aZ., 1989; Sommer et al., 1990) have not led to association of any of these genes with specific genetic diseases, although it has been speculated that defects in GABA, subunits might lead to seizures (Meldrum, 1989) or to psychiatric disorders (Hebebrand and Friedl, 1987). We have recently mapped the GABRB3 gene to human chromosome 15qllq13, a region frequently deleted in two genetic syndromes, Angelman syndrome and PraderWilli syndrome (Wagstaff et al., 1991). This localization of GABRB3 and the role of GABA in neuronal inhibition suggest a possible involvement of this gene in Angelman syndrome, a disorder characterized by seizures, severe developmental delay, disordered movements, and inappropriate smiling and laughter (Angelman, 1965). We have isolated and sequenced a human cDNA for the /33 subunit and have found that, as with other members of this receptor family, there is a very high degree of evolutionary conservation of amino acid sequence. Most amino acid substitutions are found within a large intracellular domain of the protein. The functions of this intracellular region are not known, although a recognition site for protein kinase A present in all /3 subunits suggests that at least part of the large intracellular domain may be involved in regulation of chloride channel function (Schofield et al., 1987; Porter et al., 1990). We have mapped Gabrb-3 in the mouse to proximal chromosome 7, between Tam-l and Mtv-1, approximately 1 CM proximal to Mtv-1 (Fig. 4). Two other markers from human 15qllq13, DN34 and DNlO, have also recently been mapped to this location (loci D7Hmsl and D7Nic1, respectively), and there are no recombinants among 26 BXD recombinant inbred strains between Gabrb-3, D7Hms1, D7Nic1, and the xenotropic virus locus Xmv-33 (Chaillet et al., 1991; Nicholls, 1989). This localization of Gabrb-3 provides further evidence for a region of conserved synteny between human chromosome 15qllq13 and mouse chromosome 7. Examination of the mouse genetic map shows a neurological mutation in this region, twt (twister) (Lane, 1981). This is a recessive mutation that causes abnormal head movements, circling, and inability to swim, and is located t4 CM from p (pinkeyed dilution), which lies between Tam-l and Mtv-1.

AND

LALANDE

Also, homozygotes for some radiation-induced mutations at the p locus show abnormal movements (e.g., p”; Hollander et al., 1960). In the human, maternal deletions of 15qlIq13 are associated with Angelman syndrome, while paternal deletions of this region are found in Prader-Willi syndrome, whose phenotype is very different. These different phenotypes associated with different origin of deletion suggest that one or more genes within this region show genetic imprinting, i.e., differential expression from maternal and paternal chromosomes. Extensive studies of imprinting effects associated with mouse chromosome 7 have been conducted using Robertsonian and reciprocal translocations. These studies have indicated that two regions of chromosome 7 show definite imprinting effects (Searle and Beechey, 1990). Maternal duplication/paternal deficiency for chromosome 7 distal to the T(7;15)9H breakpoint results in intrauterine growth retardation, small placentae, and antenatal death; maternal duplication/paternal deficiency for the smaller region distal to the T(7;18)50H breakpoint also produces antenatal death. Paternal duplication/maternal deficiency for the region distal to the T50H breakpoint results in early embryonic lethality. Maternal duplication/paternal deficiency for the region proximal to the T9H breakpoint leads to prenatal growth retardation and neonatal death. Paternal duplication/maternal deficiency for chromosome 7 proximal to the T50H breakpoint has no apparent effects on viability or phenotype. These results are summarized in Fig. 4. The T9H breakpoint lies approximately 1.5 CM proximal to the p locus (Searle, 1989). Gabrb-3 is likely to lie distal to p and therefore distal to the T9H breakpoint, although this localization will need to be confirmed either by linkage studies or by in situ hybridization to translocation chromosomes. Gabrb-3, as well as the other loci defined by probes from human 15qllq13 (D7Hmsl and D7Nicl), is therefore within a region for which no imprinting effect has been seen for paternal duplication/maternal deficiency and for which the imprinting status is not clear for maternal duplication/paternal deficiency. Mice with paternal duplication/maternal deficiency for chromosome 7 proximal to the T50H breakpoint should provide an ideal experimental system in which to test for differential expression of Gabrb-3 and other genes from human 15qllq13 on maternally versus paternally inherited chromosomes. Examination of the effects of maternal duplication/ paternal deficiency of the region of mouse chromosome 7 homologous with human 15qllq13 will be more difficult, due to the lethality associated with

GABRB3

maternal proximal

cDNA

duplication/paternal deficiency and distal regions of chromosome

MAPS

TO

MOUSE

of both 7.

(1960). Pleiotropic X-irradiated mice.

1.

ANGELMAN, cases. Dev.

2.

BATESON, A. N., HARVEY, R. J., BLOKS, C. C. M., ANDDARLISON, M. G. (1990). Sequence of the chicken GABA, receptor 83 subunit cDNA. Nucleic Acids Res. 18: 5557. 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.

3.

H. (1965). Med. Child

“Puppet Neural.

children”: 7: 681-688.

A report

BUTLER, M. G., AND PALMER, C. G. (1983). Parental chromosome 15 deletion in Prader-Willi syndrome. 1285-1286.

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CHAILLET, J. R., KNOLL, J. H. M., HORSTHEMKE, B., AND LALANDE, M. (1991). The syntenic relationship between the critical deletion region for the Prader-Willi/Angelman syndromes and proximal mouse chromosome 7. Genomics 11: 773-776. CU?TING, G. R., Lu, L., O’HAR.A, B. F., KASCH, L. M., MONTROSE-RAFIZADEH, C., DONOVAN, D. M., SHIMADA, S., ANTONARAKIS, S. E., GUGCINO, W. B., UHL, G. R., AND KAZAZIAN, H. H., JR. (1991). Cloning of the y-aminobutyric acid (GABA) pl cDNA: A GABA receptor subunit highly expressed in the retina. Proc. Natl. Acad. Sci. USA 88: 26732677. DAVISSON, M. T., AND RODERICK, T. H. (1989). Linkage map. Zrz “Genetic Variants and Strains of the Laboratory Mouse” (M. F. Lyon and A. G. Searle, Eds.), 2nd ed., pp. 416-427, Oxford Univ. Press, Oxford. FJZINBERG, A. P., AND VOGELSTEXN, B. (1983). A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 132: 6-13. FRANKEL, W. N., STOYE, J. P., TAYLOR, B. A., AND COFFIN, J. M. (1989). Genetic analysis of endogenous xenotropic murine leukemia viruses: Association with two common mouse mutations and the viral restriction locus Fv-1. J. Viral. 63: 1763-1774.

6.

7.

8.

9.

10.

11.

origin Lancer

of 1:

HEBEBF~AND, J., ANLI FRIEDL, W. (1987). Phylogenetic receptor research: Implications in studying psychiatric and neurological disease. J. Psych&r. Res. 21: 531-537. HOLLANDER, W. F., BRYAN, J. H. D., AND GOWEN, J. W.

at the p locus

from

13.

KIRKNJZSS, E. F., KUSIAK, J. W., FLEMING, J. T., GOCAYNE, J. D., AND VENTER, J. C. (1991). Isolation, characterization and localization of human genomic DNA encoding the 81 subunit of the GABA, receptor (GABRBl). Genamics 10: 985 995.

14.

KNOLL, J. H. M., NICHOLLS, R. D., MAGENIS, R. E., GRAHAM, J. M., JR., LALANDE, M., AND LA?T, S. A. (1989). Angelman and Prader-Willi syndromes share a common chromosome 15 deletion but differ in parental origin of the deletion. Am. J. Med. Genet. 32: 285-290. LANE, P. W. (1981). New mutants. Mouse News L.&t. 64: 59.

16.

LEDBEITER, D. H., RICCARDI, V. M., AIRHART, S. D., STROBEL, R. J., KEENAN, B. S., AND CRAWFORD, J. D. (1981). Deletions of chromosome 15 as a cause of the Prader-Willi syndrome. N. Engl. J. Med. 304: 325-329.

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LOLAIT, S. J., O’CARROLL, A.-M., KUSANO, K., MULLER, J.-M., BROWNSTEIN, M. J., AND MAHAN, L. C. (1989). Cloning and expression of a novel rat GABA, receptor. FEBS L&t. 246: 145-148.

18.

MAGENIS, R. E., BROWN, M. G., LACY, D. A., BUDDEN, S., AND LAFRANCHI, S. (1987). Is Angelman syndrome an alternate result of de1(15)(qllq13)? Am. J. Med. G&et. 28: 829838.

19. 20.

Deleted in proof. MELDRUM, B. S. (1989). pathogenesis and treatment eel. 27: 3s-11s.

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NICHOLLS, R. D. (1989). A cDNA mosome 15q maps near Mtv-1 Mouse News L&t. 84: 87-88.

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NICHOLLS, R. D., KNOLL, J. H. M., BUTLER, M. G., KARAM, S., AND LALANDE, M. (1989). Genetic imprinting suggestedby maternal heterodisomy in nondeletion Prader-Willi syndrome. Nature 342: 281-285.

23.

OLSEN, GABAA

24.

PORTER, N. M., TWYMAN, R. E., UHIZR, M. D., AND MACDONALD, R. L. (1990). Cyclic AMP-dependent protein kinase decreases GABA* receptor current in mouse spinal neurons. Neuron 5: 789-796.

25.

SAMBROOK, J., FRITSCH, E. F., AND MANIATIS, T. (1989). “Molecular Cloning: A Laboratory Manual,” Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.

26.

SCHOFIELD, P. R., DARLISON, M. G., FUJITA, N., BURT, D. R., STEPHENSON, F. A., RODRIGUEZ, H., RHEE, L. M., RAMACHANDRAN, J., REALE, V., GLENCORSE, T. A., SEEBURG, P. H., AND BARNARD, E. A. (1987). Sequence and functional expression of the GABAA receptor shows a ligand-gated receptor super-family. Nature 328: 221-227.

27.

SEAR=, A. G. (1989). Chromosomal variants: Numerical variants and structural rearrangements. In “Genetic Variants and Strains of the Laboratory Mouse” (M. F. Lyon and A. G. Searle, Eds.), 2nd ed., pp. 582-616, Oxford Univ. Press, Oxford.

28.

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on three

4.

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KAPLAN, L. C., WHARTON, R., ELIAS, E., MANDELL, F., DONLON, T., AND LAG, S. A. (1987). Clinical heterogeneity associated with deletions in the long arm of chromosome 15. Am. J. Med. Genet. 23: 45-53.

15.

REFERENCES

1077

7

12.

ACKNOWLEDGMENTS We thank L. M. Kunkel (Children’s Hospital, Boston, MA) for a human fetal brain cDNA library, John Fleming and Craig Venter (National Institutes of Health, Bethesda, MD) for PCR primers, and Peter D’Eustachio (New York University) for Chinese hamster/mouse somatic cell DNA specimens. We also thank Joan Knoll for helpful comments on the manuscript. This research was supported by NIH Grant HD18658. J.W. was supported by NIH Training Grant T32GM07748. J.R.C.‘s work was performed in the laboratory of Dr. Philip Leder, and was supported by a grant from E. I. DuPont de Nemours Co., Inc.

CHROMOSOME

R. W., AND TOBIN, receptors. FASEB

GABAergic of epilepsy.

mechanisms in the Br. J. Clin. Pharma-

from proximal human chroon mouse chromosome 7.

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The GABAA receptor beta 3 subunit gene: characterization of a human cDNA from chromosome 15q11q13 and mapping to a region of conserved synteny on mouse chromosome 7.

A cDNA encoding the human GABAA receptor beta 3 subunit has been isolated from a brain cDNA library and its nucleotide sequence has been determined. T...
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