GENOMICS

9. 3%-:-I%

(1991)

SHORT COMMUNICATION The cDNA Sequence and Chromosomal of the Murine GABA, GUI Receptor W. J. KEIR,*,’ *Department

C. A. KOZAK,t

A. CHAKRABORTI,

AND J. M. SIKELA*

of Pharmacology, Alcohol Research Center, University of Colorado Health Sciences Center, Denver, Colorado and tLaboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892 Received

June

12,

1990,

revised

5~.1991 Academic

Press. Inc.

The GABA,/BZ receptor is a member of a gene superfamily of ligand-gated ion channels that includes the nicotinic acetylcholine receptors and the strychnine-sensitive glycine receptor (Changeux et al., 1987; Grenningloh et al., 1987; Schofield et al., 1987; Betz and Becker, 1988). Several distinct classes of subunits or polypeptide types of the GABA,/BZ receptor have been identified and are designated N, 8, y, and 6 (Schofield et al., 1987; Levitan et al., 1988; Lolait et al., 1989; Pritchett et al., 1989; Khrestchatisky et al., 1989; Shivers et al., 1989; Malherbe et al., 1990). The members of each subunit class have about 70% amino acid sequence identity to each other, whereas there is only about 30% homology among ’ To whom correspondence should he addressed at Department of Pharmacology, Alcohol Research Center, Box C-236. ~lniversity of’ Colorado Health Sciences Center, 4200 East 9t.h Avenue. Denver, CO 80262. U888~75U/91 %H)O Copyright ‘cm 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.

October

10.

80262;

1990

members of different classes. Although the subunit struct.ure of this receptor has not yet been defined, there are data that suggest that different combinations of subunits produce physiologically distinct receptors (Levitan et al., 1988; Pritchett et al., 1988, 1989; Blair et al., 1988; Shivers et al., 1989; Sigel et al., 1989; Malherbe et al., 1990). Differential distribution of GABA, receptor subunit transcripts in the brain have been reported (Wisden et al., 1988, 1989a,b,c; Siegel 1988), suggesting that GABA, receptor complexes in different regions of the brain are composed of different subunits. The al transcript is more abundant than other (Ysubunit transcripts and is found in layers II-IV of frontal cortex, the cerebellum, the olfactory bulb, the inferior colliculus, and the substantia nigra (Levitan et al., 1988; Siegel, 1988; Wisden et al., 1988, 1989c; Khrestchatisky et al., 1989). These brain regions with a high al mRNA content also show a striking correlation with the distribution of type I rather than the type II GABA,/BZ receptors, which are distinguished by their responses to benzodiazepines and P-carbolines (Squires et al., 1979; Wisden et al., 1989c; Olsen and Tobin, 1990). The GABAJBZ receptor is of particular interest because it contains binding sites for several classesof pharmacologically active drugs that act allosterically with the GABA agonist site or the receptor channel. These include anticonvulsant (barbiturates), convulsant (picrotoxin), anxiogenic (@-carbolines), and anxiolytic (benzodiazepine) agents (Braestrup et al., 1982; Olsen, 1982; Barker et al., 1984; Olsen and Venter, 1986). In addition, recent behavioral (Liljequist and Engel, 1982; Frye and Breese, 1982; Martz et al., 1983) and electrophysiological and neurochemical (Mereu and Gessa, 1985; Suzdak et al., 1986; Allan and Harris, 1986) observations have implicated the GABA,/BZ receptor chloride ionophore system in

The murine GABAJbenzodiazepine (GABAJBZ) reeeptor crl subunit cDNA has been isolated from a BALB/c mouse brain library and sequenced. The cDNA is 2665 nucleotides long with an open reading frame of 455 amino acids. It shows significant homology to the GABA, receptor al subunit cDNA sequences of other species. Excluding deletions, the murine GABA, crl receptor exhibits 96% nucleotide and 100% amino acid sequence homology to the rat al receptor cDNA and over 91% nucleotide and 98% amino acid sequence homology to the bovine and human (~1 receptor cDNAs in the protein coding region. This murine cDNA was used to locate the (~1 receptor subunit gene, Gabra-1, to murine Chromosome 11 between R-3 and Rel. This assignment extends proximally the segment of mouse Chromosome 11 with known homology to human chromosome

5.

t R. A. DEITRICH,*

Location Gene

390

:m GAIlTTCCGCTACAGAGATGGGGATTAGGGCCAGAGTGCAAGTTAIVLTTGCGCTGCATATAA~TGGGCGGAT~GGTGTCCAGATCcAGTGGC?(;T:‘AC:‘ 13?-

A(‘(‘TTCCTTTCTRAMTAAAATCTCTCTGGCATGMGTCACCGCCTATTTCACATCCGGTTTGCGCTGGGACGTAT'rAC~ACT[;TCTTG~TACAGAG~~A~

:,?m

CT'?TGT_GTATAGCTGCAGATTGGATATTGGGAAGCAAATTTGGGTATGAAATCTCCAGTGCAGGAGCACGCAGAGTCCATGRTGCCTCAGACCI;Z(;:(;I\

X04-

GTGAGCGCGGCGCGAGGACGCCCTCCGCCCGGCGC

r,J,m

TCTGGAGCGATCCGGTGCCCAGAGGGGGCCCCGAGCTGTGC~GCCCG~G Leu Ser Thr CTG AGC ACA

TCCCGCGTCGCAGACTCGCGCAGCTCCGACTCACCGCGATCCTC~CTCCCACAC~T'rTC?CCCC(;(;

v no ’ * *

q

Trp TGG

Asn AAC

?hr TYr ACC ACT

Met Lys Lys Ser Arg Gly Leu Ser Asp Tyr LEU ATG AAG AAA AGT CGG GGT CTC TCT GA': TAT CT:

Le" Ser Gly Arg Ser Tyr Gly Gin Pro CTG TCG GGA AGA AGC TAT GGA CAG CCC cl

Ser

Gin TCC CM

3

Asp Glu GAT GM

Leu CT:

Lys Asp AAA GA:

5 i i-"

4 5 6 Val F'he Th; Arg Ile Leu Asp Arq Leu Leu Asp Gly Tyr Asp As" Arg Leu Arg Pro Gly Ir?l: Gly Glu Ar,; VdI -h: G?.: GT(' TTC ACA AGA ATT TTG GAC CGA CTG CTG GAC GGT TAT GAC AAT CCT CTC AGA CCA GGT TTG GCA GAG CGT GYA ACT GM

$51-

V.ZIl Lys Thr G?: AAG ACC

134m

Gin Ser Trp Lys Asp Glu Arg Leu Lys Phe Lys CAP AGT TGG AAG GAT GAA AGA TTA AAA TTC AAA

Asp I?e Phe Val Thr GAC ATT TTC GTC ACC

Thr Pro Asp Thr ACT CCA GAT ACA

Sfr Phe Gly Pro Val Ser Asp His Asp Met S:u :yr AGT TTC GGA CCA GTT TCA GAC CAC GAT ATG GAG :‘A-: Gly Pro Met Thr Val Leu Arq Leu Asn GGA CCC ATG ACA GTG CTC CGG CTA AAC

Phe Phe His Asn Gly Lys Lys Ser Val Ala His Asn TTT TTC CAC AAT GGA AAG AAG TCT GTG GCC CAC AAC

896

Thr ACA

R7!-

Asp Ala His Ala Cys Pro Leu Lys GAT GCC CAT GCC TGC CCA CTA AAA

Thr Ile Asp Val Pne Phe ACA ATA TAT GT1.; TTT 'I":C

Arg :,;'r

Asn MC

I:@ RTC

Lel: Met CTT A-G

Al.3 SCr 'yi: CCC ACT AM

Met Thr Met Pro Asn Lys Leu L~L Arg Ile ATG ACC ATG CCT AAT AAG CTC CTG CGT ATC

Glu Asp Gly Thr Leu Leu Tyr Thr Met Arg Leu Thr Val Arq Ala Glu Cys Pro Met 'iis Lel: Glu Asp Phe GAG GAT GGC ACT CTG CTG TAC ACC ATG AGG TTG ACC GTG AGA GCT GAA TGC CCA ATG ICAC TTG GAA GAC TX Phe Gly Ser Tyr Ala Tyr Thr Arg Ala Glu Val Val 'Fyr Glu Trp Tnr Arg TTC GGA AGC TAT GCT TAT ACA AGA GCA GA?, GTT GTC TAT GAG TGG ACC ACA

lSiB-

Ala XC

Arq Ser Val Val Val Ala Glu Asp Gly Ser Arq Leu As" CGT TCA GTG GTT GTA GCA GAA GAT GGG TCA CGT TTA AAC

:139-

va1 Gin Ser Ser Thr Gly Glu GTT CAG TCC AGT ACT GGA GM

1;20-

Thr ACA

: iOl-

Val Pbe Gly Val Thr Thr Val Le" Thr Met Thr Thr STC TTT GGA GTG ACG ACT GTT CTG ACT ATG ACA ACC

:3H:-

Thr ACA

iiihi-

Thr Lys Arg Gly Tyr Ala Trp Asp Gly Lys ACC AAG AGA GGG TAT GCG TGG GAT GGC AAA

1:44-

Asn MC

:621-

Ser Ala Thr Ile Glu Pro Lys AGT GCG ACC ATA GAA CCG AAA

17C6-

Ile A?C

:187-

GIG Pro Gln Leu Lys Ala Pro Thr GAG CCT CAG CTA AAA GCC CCC ACA

Gln Tyr Asp Leu CAG TAT GAC CT-

Leu Gly Gin Thr CTT GGA CAA ACA

Tyr Val Val Met Thr Thr His Phe His Leu Lys Arq Lys TAT GTG GTT ATG ACG ACT CAC TTC CAC TTG AAG AGA AAA

Yrc CC:

Met ATG

Cli: Pro SAS ZcA

Val Asp 5er Gly Ile GTT SAC TC? GSA AT-

1le Gly Tyr Phe Val Ile Gin ATT GGC TAC TTT GTT ATT CAA

Tyr Leu PRO Cys Ile Met Thr Val Ile Leu Ser Gin Val Ses Phe Trp Leu Asn Arg Glu Ser Val Pro Ala A:g Tnr TAT CTG CCG TGC ATA ATG ACA GTT ATT CTC TCC CAA GTC TCC TTC TGG CTC AAC AGA GAG TCA GTA CCA GCA AGA ACT Leu Ser Ile Ser Ala Arg Asn Ser Leu Pro Lys Val Ala Tyr A;a TTG AGT ATC AGT GCC AGA AAT TCC CTC CCG AAG GTC GCT TAT GCA

Ala Met Asp Trp Phe Ile Ala Val Cys Tyr Ala Phe Val Phe Ser Ala Leu Ile Glu E'hc Ala Tkr Va: Asn GCT ATG GAC TGG TTT ATT GCA GTA TGC TAT GCC TTT GTT TTC TCA GCT CTG ATT CAG TTT GCC ACA GT? AA;

Asn AAC

"hr ACA

Tyr Ala Pro Thr Ala Thr TAT GCT CCT RCA GCA ACC

Ser Val Val Pro Glu Lys Pro Lys Lys AGC GTG GTT CCA GAA AAG CCA AAG AM

Ser Tyr Thr AGC TAT ACC

Pro As" CCT AAC

Glu Val Lys Pro Glu Thr GAA GTC RAG CCT GAG ACA

Lys Pro Pro AAA CCA CCA

Glu Pro Lys Lys GAA CCC AAG AAA

Thr Phe Asn ACC TTT &AC

Leu Val Tyr Trp Ala Thr TTA GTC TAT TGG GCC ACT

I'hr :::

I.ks /,iiG

by.; ,SA/+

Ala Lys SCT AM

Ser Val Ser Lys AGC GTC AGC AAA Tyr L~L Asn TAT TTA AA:

Arq AGA

GTTCTTTTAGTCGTATTCTG-TGT"CAGTCCTCTGCAGTCCTCTGCACCGAGM'~~GC~~

?974-

TGTCTGACAGTCCAGAGCAGAGCAGAGTATTCAGCTCAGGGACAGGATTC~GAGAGG~GCCAGGGAGCAA~GCATGTCA~ACGGAGATAGACAAGAGGA

2075-

AGACAGAGGGTAAGAAGGTCCAAAGATAGGAGAAAGTAG

?i76-

CTAAAAAAATATATACGTCAAARATATATTTTTGAAAGGCACAA

2277-

ATGATATAGTGTGCTTATGTTTTTATTCGTCAATGTTGAAGCTGATATATAGATTT~TGTTTTGTTTGTC~ATT~~ATTCCCTAGGCTTTC'rCTTTT

2479-

ATCCAGTGTGGGGAAACCCTTTCAATCGGGGCTACACTGCTGTCATCTGAACTTTTACCAGTAGACTCTATAGAGATCAGCCAGCC?AACACAGA?GTTTAC

2580-

TTGATAGAATCTGAAATAAATAAAAGAATGGAATGGAATAATTT~CTATCCTTTTTACTCTTAT~CCCAGATAGCCCATTCACCCAA

FIG. 1. DNA sequence and deduced amino acid was sequenced according to the Sanger dideoxy DNA employed to produce unidirectional nested deletions prepared from colonies using alkaline minipreps and universal primer. ‘The DNA sequence of the opposite cleotides as primers. The sequences of these primers

:.e A-7

Leu Ala Arg Gly Asp Pro Cly Leu Ala Thr Iie TTA GCC AGG GGT GAC CCC CGC TTG GCA ACT AT"

Asp Arq Leu Ser Arq Ile Ala Phe Pro Leu Leu Phe Gly Ile Phe Asn GAC CGA CTG TCA AGA ATA GCC TTT CCG CTG CTA TTT GGA ATC TTT MC Pro His Gin AM* CCC CAT CAA TAG

Val Lys As? Pro Leu GTA AAG GAT CCT CT:

Tyr I'iiT

AAAAAACCAGAGCTTRACTGTAACCACAGAGTCATTTGTAGATATATAT?TCCA~TAT~

2665

sequence of the murine GABA, receptor ~1 subunit cDNA. The isolated mouse cDNA sequencing procedure (31). The Exe/mung DNA sequencing system (Stratagene) was of the mouse GARA, receptor cDNA. The resulting shortened DNA clones were used to derive the complete cDNA sequence of the clone from one strand using the Ml3 strand was obtained using the Ml3 reverse primer and 10 synthetic antisense oligonuwere based on the cDNA sequence obtained from the first strand and each primer was 391

392

SHORT

COMMUNICATION

the actions of ethanol. As a first step in establishing whether GABA,/BZ receptor genes difl’er among various mice known to differ in their ethanol sensitivity, we have begun to isolate various mouse GABA,/ BZ receptor subunit genes. In this report, we describe the molecular cloning and sequencing of the murine GABA, receptor cul subunit cDNA and the chromosomal mapping of this gene. A 40-bp oligonucleotide. 5’.GAAGAAAAGTCCGGGTCTCTCTGACTACCTTTGGGCCTGG-3’, corresponding to the most 5’ end of the protein coding region of the published bovine GABA, recept.or ~1 subunit cDNA (Schofield et al., 1987), was labeled with T4 polynucleotide kinase using [r-“‘P]ATP and used to screen a Xgtll BALB/c mouse brain cDNA library (size selected for inserts greater than 2 kb) by plaque hybridization as described by Maniat.is et al. (1982). The library was kindly provided by Yoav Citri. From 1 X 106plaques, one clone containing an insert of 2.66 kb was isolated. Phage DNA from this plaque was cut with EcoRI and the cDNA insert ligated into Bluescript plasmid (Stratagene, LaJolla. CA) and sequenced according to the Sanger dideoxy DNA sequencing procedure (Sanger et al., 1977) using the Exe/mung sequencing system of Stratagene. The nucleotide sequence of the clone contains a 1365.nucleotide (455 amino acids) open reading frame preceded by 454 bp in the 5’ untranslated region and followed by 846 bp in the 3’untranslated region. There appears to be no polyadenylation signal in the last 70 bp of the 3’ untranslated sequence (Fig. 1). The mouse GABA, cul receptor sequence was compared with the sequences for this receptor in rat (Lolait et al., 1989; Mahan, personal communication), bovine (Schofield et al., 1987), and human (Pritchett et al., 1988; Schofield et al., 1989) and this comparison revealed considerable homology. The base pair homology of t,he coding region of this mouse gene, excluding deletions, is 96% (1304/1365 bp) with respect to rat, 91% (1242/1365 bp) with respect to bovine, and 91.5% (1249/1365 bp) with respect to human. The amino acid homology, excluding deletions, is 100% with respect to rat, over 99% (4521455) with respect, to bovine, and over 98% (448/455) with human sequences (Fig. 1). The mouse and rat cDNA sequences lack the bovine and human leucine 31 (Fig. 1). All t,hree of the amino acid subst.itutions that differentiate the mouse and bovine cDNA sequences are located in the signal sequence. Excluding the signal sequence, the nonhuman genes are identical in amino

acid sequence and dither from the human sequence by one amino acid. To map the gene encoding the GAHA, receptor tul subunit to a specific mouse chromosome, we analyzed a panel of Chinese hamster x mouse somatic cell hybrids by Southern blot analysis. The mouse cul cDNA receptor probe cross-hybridized with hamster Hind111 fragment,s of 8.9, 5.7, 4.0, and 3.4 kb; corresponding mouse Hind111 fragments were 14.5, 12.8, 11.3, 3.6, 2.9, and 1.7 kb (Fig. 2A). The Chinese hamster parental DNA digested with XbaI showed 3 bands of 8.2, 5.9, and 4.7 kb, while the mouse parental DNA digested wit,h XhaI showed 5 bands of 14.9, 10.7,5.8.4.2, and 1.7 kb (figure not shown). None of the hybrids contained Hind111 or XbaI mouse GABA, tul fragments. Examination of the mouse chromosome cont.ent of these hybrids revealed a correlation with mouse Chromosome 11 (data not shown). All 29 of the hybrids lacked Chromosome 11, whereas all other mouse chromosomes were present in at least two of the independent hybrid lines tested. These data support the assignment of the gene for the ~1 subunit of the murine GABA, receptor (&bra-1) t.o Chromosome 11. To define a more precise location for Gabm-1 on Chromosome 11, DNAs extracted from the parent,al mice of an intersubspecies backcross were examined by Southern blot analysis for restriction fragment length polymorphisms (RFLPs). DNAs from the inbred strain NFS/N and wild-derived Mus musculus mice digested with EcoRI produced different restriction fragments reactive with the GABA, receptor cDNA. NFS/N mouse DNA produced four EcoRI bands of 11, 5.7, 4. and 3.5 kb, while M. m. muse&s produced four bands of 1I, 5.7,4, and 2.9 kb (Fig. 2B). DNAs of 67 of 109 mice of the backcross (NFS/N ‘x M. m. musculus)F, x M. m. musculus produced the 3.5-kb EcoRI fragment consistent, with the expected 1:1 segregation ratio for a single gene. The backcross mice were also typed by Southern blot analysis for the inheritance of RFLPs of the Chromosome 11 markers 11-3and Rel. Examinat,ion of the backcross mice for the inheritance of these different polymorphisms showed Gabra-1 to he linked to Rel and 11-3 (Table I). The data imply the following gene order: centromere-Rel-(9.9 -+ 3.1 cM)-Gnbra-Z(3.3 t 2.3 CM)-f1-:3. Buckle and colleagues (1989) have localized the human homolog ofthis gene, GABRAl, to chromosome 5 (bands q34-q35), which is known to share extensive

approximately 2.50 hp from the next. The putative signal sequence cleavage site is indicated hy an arrow. The tour putative membrane spanning hydrophobic domains are indicated by overscoring solid bars. Amino acid changes between mouse and hovine or human GABA, cDNA sequences are boxed. Numbers helow each box correspond to the following amino acid changes: 1. Arg (human): 2. Pro (hovine and human); 3. Cys (human); 4, Ile (human): 6, Phe (hovine) or Leu (human); 6. Thr (human and hnvine); ‘i, Arg (human). The asterisk (*) denotes the position of the hovine and human leucine 31 that is deleted in the mouse and rat (:ABA, rDNA sequences. The two cysteine residues in the putative extracellular domain thought to form a disulfide bridge are highlighted hy solid circles. The invariant proline residue in the first transmemhrane region found in GABA, receptors and other receptors coupled to ion channels (4) is in hold capital lettering.

SHORT

a

b

c

393

COMMLTNICATION

TABLE

d

14.5 12.8 11.3

1

Segregation of the Gabra-1 Hybridizing Restriction Fragment with Alleles of Rel and R-3 in 91 Progeny of an Intersubspecies Backcross

8.9-

Inheritance NFS/N

Kc1

Mice

of the allele” -__

Chhra-

I

11L.3

Number of mice 311 “0

Nonrecomhinants

+

+

4

Single

t

+ -

t

2 I) I>

1

4

1

3.6 2.9

recombinants

+

Recombination* a

b

cdef

Percentage

gh

6

FIG. 2. DNAs from mouse livers, Chinese hamster cells, and somatic cell hybrids were digested with restriction enzymes. run on agarose gels. and transferred to nylon membranes. The filters were hybridized with the ‘UP-laheled GARA, LYI receptor cDNA probe which represented the 2665.1,~ EcoRI insert from pBluescript (>106 cpm/ml). (A) Southern blot analysis of HindIII-digested mouse and Chinese hamster k mouse hybrid DNAs using the murine GARAA c,l cDNA as prohe. Lanes a-c, independent somatic cell hybrids; lane d, NFS/N mouse. Fragment sizes in kilohases are indicated to the right for mouse and for Chinese hamster to the left. Twenty-nine hamster ‘X mouse somatic cell hybrids were produced hy fusion of the Chinese hamster cell line E36 with cells of various inhred mcouse strains as described previously (15). The mouse chromosome content of 11 hybrids was determined by Giemsa-trypsin banding; 18 hybrids were typed for markers on specific chromosomes. (B) Southern blot analysis of EcoRI-diBested backcross DNAs using the murine GABA, nl cDNA as prohe. Lanes a-g, independent backcross mice; lane h is NFS/N mouse. Fragment sizes in kilohases are indicated to the right for NFS/N and to the left for M. m. musculus. NFS/N strain mice were obtained from the Division of Natural Resources, NIH, Bethesda, Maryland, and C58/d mice from the Jackson Laboratory, Bar Harbor, Maim?. hf. ni. musculu.~ (Skive) mice were provided hy Dr. M. Potter from his colony and maintained at Hazelton Laboratories, Rockville. Maryland (NC1 Contract NOl-CB-71805). NFS/ N and C58/J females were mated with M. tn. musculu.s males and the F, females were backcrossed with M.m. musculu.s males to produce the experimental animals.

Locus

pair

r/n

(il

recombination SE)

” II-:3 was typed following E:coRI digestion using the cDNA clone pIL-3 ( 16). Rrl was typed following Hind111 digestion using pHHS rel-1 (7) kindly provided by Dr. N. Rice (NCI, NIH). For 11-3, NFS/N mouse DNA produced a IO.“-kb EcnRI fragment, whereas M. m. muscul~s DNA produced an 8.4.kb EcoRI fragment. For Re1. the fragment sizes following Hind111 digestion were 19.0 kh for NFS/N and 13.5 kh for M. m. mu.sculu.s. b Percentage recombination between restriction fragments and standard error (SE) were calcldated according to (ireen (11) from the number of recombinants (r) in a sample size of n. ’ An additional 30 mice were tUyped only for Rcl and (hbra-I for a total percentage recombination of I)/91 = 9.9 f 3.1.

homology wit.h mouse Chromosome 11 (Nadeau, 1989). The mapping of the Gabra-1 locus to mouse C,hromosome 11 extends this region of linkage homology by providing the most centromeric locus in this homology group on mouse Chromosome 11. Only one other GABA,/BZ receptor subunit gene has been mapped in mice. This gene, Gabra-3, encodes a different N subunit and was located on the murine X chromosome (Buckle et nl., 1989). Genes for the related nicotinic acetylcholine receptor have also been under investigation and a recent st,udy has mapped one of these, the 13subunit gene, to mouse Chromosome 11 (Heidmann et al., 1986). Its map location is, however, clearly distal to that of Gabra-I, suggesting that these genes are not part of the same gene complex. Given the importance of t.he GABA,/BZ receptor in mediating central nervous system responses, the mouse chromosomal location of the GABA, receptor tul subunit cDNA is important in determining its po-

394

SHORT

COMMIJNICATION

tential role in previously described mutants involved in CNS function. Several studies support the hypot,hesis that genetic differences in ethanol and benzodiazepine sensitivities are associated with GABA, receptor gene function. Specifically, it has been suggested that genetic differences in acute ethanol and diazepam sensitivities are associated with changes in the GABA,/BZ receptor Cl- channel ionophore complex (see Harris and Allan, 1989, for review). DiEerences in ion channel function have also been shown in several mouse lines specifically bred to display genetic differences in ethanol intoxication (Allan and Harris, 1986) and benzodiazepine sensitivity (Allan et al., 1988). While the number and chromosomal distribution of the genes controlling these differences have not been ascertained, it is possible that a defect in the Gabra-1 sequences could contribute to such ditierences in alcohol and drug sensitivity. However, the region of mouse Chromosome 11 containing Gabra-1 has currently no known mutants that could be ascribed to a defect in Gabra- 1. Because data indicate that this receptor complex is involved in ethanol actions and the actions of other drugs, we are now investigating the structure and function of the GABA,/BZ receptor genes in mouse lines with differential sensitivities to ethanol and other drugs. The cloning, sequencing, and genetic mapping of the murine al cDNA are the first steps in the analysis of the genetic diversity and differential expression of this family of genes in the mouse.

AND PETERSON. E. N. (1982). Interaction of convulsive ligands with henzodiazepine receptors. Science 216: 12411243. 7.

BROWNELL, E., AND O’BRIEN, S. J. (1985). Genetic characterization of human c-rel sequences. Mol. Ccl/. Biol. 5: 2826“83 1

8.

BUCKLE. V. .I., FUJITA, N., RYDER-COOK, A. S., DERRY, .J. M. J.. BARNARD, P. ,I., LEBO, R. V., SCHOFIELD, P. R., SEERIIRG, 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. Nwron 3: 647m 654.

9.

CHANGEUX, J.-P., GIRAUDAT, .J., AND DENNIS, M. (1987). The nicotinic acetylcholine receptor: Molecular architecture of a ligand-regulated ion charmeL Trends H&hem. Sri. 8: 459.. 465.

10.

FRYE, G. I).. AND BREESE, G. R. (1982). GABAergic modulation of ethanol-induced motor impairment. J. Pharmacol l?xp. Ther. 223: 7X-756.

1 I.

GREEN, E. I,. (1981). Linkage, Znt “Genetics and Prohahility ments” pp. 77 113, Macmillan,

12.

GRENNINGLOH, G., RIENITZ, A., SCHMITT. B., METHFESSEL. C.. ZENSEN, M., BEYREUTHER, K., GUNDELFINGER. D. E., AND BETZ, H. (1987). The strychnine-binding suhunit of the glycine receptor shows homology with nicotinic acetylcholine receptors. Natuw (London) 328: 215-220.

13.

HARRIS, R. A., AND ALLAN, A. M. (1989). Alcohol intoxication: Ion channels and genetics. FASER J. 3: 1689-1695.

14.

HEIDMANN, 0.. BUONANNO, A., GEOFFROY, B., ROBERT. B., (:UENET, J-L., MERLIE, .J. P.. AND CHANGEUX, .J.-P. (1986). Chromosomal localization of muscle nicotinic acetylcholine receptor genes in the mouse. Science 234: 866-868.

15.

HOGGAN, M. L)., HALDEN, N. F., BUCKLER, C. E., AND KOZAK, C. A. (1988). Genetic mapping of the mouse cmfms protooncogene to chromosome 18. J. Viral. 62: 10551056.

16.

IHLE. ,J. N., SILVER, .J.. AND KOZAK, C. A. (1987). Genetic mapping of the mouse interleukin gene to chromosome 11. J. Immunol. 138: 3051-3054.

17.

KHRESTCHATISKY. XIJ, W., -JACKSON, R. W.. AND TOBIN, GABA, receptors.

18.

LEVITAN. E. S., SCHOFIELD, P. R., BURT, D. R., RHEE, L. M., WISDEN, W.. KOHLER, M., FUJITA, N., RODRIGUEZ, H. F.. STEPHENSON, A., DARLISON, M. G.. BARNARD, E. A., AND SEEBURG, P. H. ( 1988). Structural and functional basis for GABA, receptor heterogeneity. Nature (London J 355: 76-79.

19.

LIUEQUIST. S., AND ENGEL, J. ( 1982). Effects of GABAergic agonists and ant,agonists on various et.hanol-induced behavioral changes. f?sychophnrmneo&v {BerfinJ 78: 71-75.

“0.

LOLAIT, S. .J., O’(‘ARROLL, A. M., KUSANO, K., MULLER, .J. M., BROWNSTEIN, M. J.. AND MAHAN, L. (1989). Cloning and expression of a novel rat GABA A receptor. FERS L&C.

ACKNOWLEDGMENTS We thank G. Boudle, B. Toland, and D. Wilson-Shaw for their invaluable technical assistance. This work was supported hy ITSPHS Grants AA03527 and AAOOO93.

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ALLAN, A. M., GALLAHER, E. J., GIONET. S. E., AND HARRIS, R. A. (1988). Genetic selection for benzodiazepine ataxia produces functional changes in the y-aminohutyric acid receptor chloride channel complex. Brain Res. 452: 118-126. BARKER, J. L., GRATZ, E., OWEN, D. G., AND STUDY, R. E. (1984). Pharmacological effects of clinically important drugs on the excitability of cultured mouse spinal neurons. In “Actions and Interactions of GABA and Benzodiazepines” (N. G. Bowery, Ed.), pp. 203-216, Raven Press, New York. BETZ, H., AND BECKER, C.-M. (1988). The mammalian glytine receptor: Biology and structure of a neuronal chloride channel protein. Neurochem. Int. 13: 137-146. BLAIR, L. A. C., LEVITAN, E. D., MARSHALL, d., DIONNE, V.. AND BARNARD, E. A. (1988). Single subunits of the GABA, receptor form ion channels with properties of the native receptor. Science 242: 577-519. BRAESTRUP, C., SCHMIECHEN, R.. HEEF, G., NIELSON, M..

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recombination and mapping. in Animal Breeding ExperiNew York.

M.. MACLENNAN, A. J., CHIANG, M.-Y.. M. B., BRECHA, N., QTERNINI, C.. OLSEN, A. J. (1989). A novel (Y subunit in rat brain Neuron 3: 745-753.

745--148.

“1.

MALHERBE, ,I. (;.. AND sites of gene receptor in

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