- Email: [email protected]
The GABAA receptor α6 subunit gene (Gabra6) is tightly linked to the α1-γ2 subunit cluster on mouse chromosome 11
NAa-asz
h, Jiangpillg
Gan
a7
Abstract We have established that the GABA ,, receptor q, (Grrhln6 1 and a, CGdmrl~ subunit penes are tightly linked on mouse chromosome I I by analyzing the strain distribution patterns of RFLPs for the two genes and microsatellite markers flanking these genes in 26 BXD recombinant
inbred strains. These results further demonstrate clustering of the GABA,,
receptor subunit penes on mouse chromosomes
and the synteny for these clusters between the mouse and human penomes. Kqxwz&t
GABA,,
receptor: Alphal zuhunil: Alpha6 subunit: RFLP; C57BLj6J
y-Aminobutyric acid (GABA) is the predominant inhibitory neurotransmitter ii; the mammalian central nerreceptors are the site of action vous system [36]. GABA, of several classes of therapeutically important drugs, including the benzodiazepines, barbiturates and inhalation anesthetics [27,36]. The GABA,, receptor is a heteropentamer composed of varying combinations of cr, ,8, y and S subunits. To date, 13 genetically distinct subunits have been identified in mammals, including 6 cy, 3 /3, 3 y and 1 6 species [3 I]. The electrophysiological and pharmacological properties of the GAIBA, receptor are dependent upon the subunit composition of the complex [31]. While strong evidence suggests that several isoforms of the GABA, receptor exist in vivo, the exact composition of individual GABA, receptor pentamers has yet to be determined. Many of the GABA, receptor subunit genes have been localized on human chromosomes. Most of these genes are grouped in cluPters. The CV:(GabruZ), (Y_,(Gd~rur’h PI (Gubrbl) and y, (Gczbrgl) genes are located on human chromosome 4 [4,8,33]. 4. second cluster, including the cyI (Gabrul), (Ye (Gabm6~, Pr (Grrbr172) and y2 (Gabrg2)
* Corresponding author. Department of Physiology. University of Oklahoma Health Sciences Center. PO Box 26901. Oklahoma City. OK 73190. USA. Fax: + I (405) 271-3181: E-mail: [email protected] 0169-328X/97/$17.00
and DBA/2J
genes, has been identified on human chromosome 5 [4,16,19,20.38,46]. The (Y~(Guhr-~.5), & (Gtrhrh3) and y3 (Ga1wg.Z) genes, located on human chromosome IS near the Angelmann-Prader Willi locus. comprise the third cluster [l-5,33,34]. Subunit genes not clustered with others include those for the LYE(G&Y& and 6 (G&Y!) subunits which are located on the human X chromosome and chromosome 1, respectively [2,41]. The mapped mouse GABA, receptor subunit genes are syntenic with those on the human chromosomes and are arranged in similar clusters. The Gabm2 and Gabrbl genes are located on mouse chromosome 5 [7] and the Gabrb3 and G&-g_? genes are tightly clustered on mouse chromosome 7 [7,35,43]. As in man, the Gabra3 gene is found on the mouse X chromosome [9]. Gabral and Gabrg2 genes are located on mouse chromosotne 11 [5,22]. The Gab&i gene has also been mapped to mouse chromosome 11 by interspecific backcross [37], however, its relationship to the (Y, and y, subunits was not determined. We have used restriction fragment length polymorphism (RFLP) and short sequence length polymorphism (SSLP) techniques in BXD recombinant inbred (RI) strains of mice to establish that the Gnbru6 gene is tightly linked to the G&-al and Gubrg2 genes on mouse chromosome 11. DNA from C57BL/6J and DBA/2J inbred mice and from 26 RI strains (obtained from Jackson Labs.) was
Copyright 0 1997 Elsevier Science B.V. All rights reserved.
P/ISO169-328X(96)00290-2
inbred strain: BXD recombinant inhred btrain
134
Hind III a, IBstE III a6 C57
DBA
C57
DBA
kb 23 ::t : 4.4 2.0 -
Fig. I. RFLPs for GABA, receptor (I, and q, subuni! probes in C57BL/6J and DBA/ZJ mice. Blots of C57BL/6J and DBA/?J genomic DNA digested with Hind111 were hybridized with a 1.8-kb cDNA probe of the human CABA, receptor (I , subunit. Blots of C57BL/6J and DBA/2J genomic DNA digested with BstEll were hybridized with a 264-bp cDNA probe of the mouse GABA, receptor a6 subunit.
isolated, digested with restriction enzymes, electrophoresed on 1% agarose gels and transferred to charged nylon membranes (Zeta Probe GT; Bio-Rad) [39]. The membranes were hybridized with cDNA clones for the (Y, or q, subunits random prime-labeled with [j2 P]dCTP (Promega). The probe used for the (Y, subunit was the full-length cDNA for the human (Y, subunit [14]. The
A
probe for the q, subunit was constructed by RT-PCR amplification of mRNA that encodes unique residues 322410 in the cytosolic loop between the TM3 and T regions of the C57BL/6J inbred mouse GABA, receptor LYEsubunit [21]. Genotyping of mouse microsatellites in inbred mice was performed by a modification of the method of Dietrich et al. [IO] as described by Bclknap et al. [1] using Mouse MapPair primers (Research Genetics). Products were electrophoresed on a native 8% polyacrylamide gel, stained with ethidium bromide and photographed. Linkage probability and the map position of the Gabru6 gene were determined from the strain distribution pattern of the RFLP and SSLP markers using the MAP MANAGER software [32]. A Hind111 RFLP for the LV, subunit was identified between progenitor C57BL/6J and DBA/2J inbred strains of mice (Fig. 1). Southern blots showed bands at 13. I, 2.6 and 1.2 kb in the C57BL/6J mice compared with 7.4, 5.3, 4.9, 2.8, 2.4 and 1.2 kb for the DBA/2J mice. We also observed a Nsil polymorphism in the LY,subunit between these two strains as predicted by Wang et al. [45] (data not shown). An RFLP also was identified for the ah subunit between the C57BL/6J and DBA/2J strains using BsrEII. A single band of 27.8 kb for the C57BL/6J and three bands of 19.6, 14.7 and 7.4 kb for the DBA/2J were seen on Southern blots (Fig. I). The strain distribution patterns of the (Y, and q, subunit RFLPs were characterized in the 26 BXD RI strains. The (Ye probe RFLP exhibited a distribution pattern among
BXD STRAINS 1
2
5
6
ii.8
P40-135
12.8
DllMit135
15.0
DllMit51
D D
17.1
DllMit174
Gabral
D D
la.2
DllMitZB
Ebf
25.0
I1 5 DllMi tZ3 DllMi t86
9 11 12 13 14 15 16 18 19 20 21 22 23 24 25 27 28 29 30 31 32 D D
DllMit53
D 0 D D R D
Gabra6
D D D D D 0 0
D
D D 0
Fig. 2. A: strain distribution pattern of Gabral and Gab&
D
in BXD recombinant inbred strains. B and D are generic symbols for alleles inherited from the
C57BL/6J and DBA/2J progenitors. respectively. Black (shaded) chromosomal regions representthose retaining markers from the C57BL/6J progenitor and white (unshaded) regions indic:lte those having alleles derived from the DBA/2J progenitor strain. A cross-over is indicated by break between the shaded and unshadedbars. using the MAP MANAGER
9:
partial linkage map of mouse chromosome I I was constructed from the data shown in A by minimizing cross-over events program version 2.6.5 1321.Genetic distancesare in CM. The map was anchored by assigning DI lMir2 to 5 CM [3].
RI maims i&mica8 with that Qf the a, subunjt RFLP (Fig. 2Al Using the data of EBlicPtt[12], we determined that t&c genes v ere Binked to the regionof nwia?ie ~~r(~~~~()~(~~~~e tp between IW-t-s.5 and 115 (Fit7 “&BP which is the same regio;a ?O wtlich GcrhwI had been previous8\:map@ by inter-subspecific backcross [5]. The G&r-0l and Gahrtraj geXS were flanked proximall) by DlldMir5l anJ di.siallj this Pinkage Sy by DIIMir20 and Ebf. We confirmed independently typing it51 and ~~~~~t2~. Strain distribution patterns for several other SSLP markers were characterized in the BXU R1 strains to identify markers tightly iinked to the GnBrul and G&r-lrh genes. One marker, DI lMitl74, was found to co-segregate with RFEPs for both GABA receptor genes. The assigned position of 51 IAJit13.5 I CM distal to P40-rsS and DIlilgir.53 [98] was confirmed by the observation of a single exchange: between these markers in line BXD-I (Fig. 2A.B). DllMit86 co-segregated with DilMir23 and 115 [I I]. Bur data establish that the murine Cdm~h gene is associated with the Gcrhrtrl-GdwgI cluster on mouse and G&rg-? chromosome I I. Bn man. the Gr,h/-~rl. Gtrhnh genes map to a restricted region on human chromosome 5q34-q35 [4,19.20,46]. These findings extend the synteny for this region conserved between mouse and man. The GABA, gene ercoding the ,8? subunit (G&&2) also has been mapped to the region of this cluster on human chromosome 5 [38]. The murine Cub162 gene has not yet been mapped, but is likely to be in the mouse chromosome 1i complex if synteny for this cluster of GABA, genes is maintained between man a;ld mouse. Our data from BXD RI analysis establish that the Gdwul and Gtrhw6 genes are likely to be within I CM (O-4 CM, 9.5% confidence limit) of one another (the limit of the resolution of these strains [40]). Wilcox et al. [46] showed that human Gahral and Gdwg.? are within 200 kb of one another. A precise c’
positioning of the human Ga61~6 gene relative to these two has not been reported. The functional significance of the clustering of these GABA, receptor subunit genes is unknown. One proposal is that clustered subunit genes may be coordinately regulated at the transcriptional level [46]. Additional molecular genetic data in both man and mouse is needed to determine
if the linkage of these genes is sufficiently close to allow co-regulation of their expression by common transcriptional regulatory sequences. The high degree of co-expression of transcripts for the lyi, & and -yz subunits among brain regions supports this idea [25,47]. However, other data suggest that independent regulation of these genes occurs. The restriction of cr6 transcription to only two regions of the mouse brain, the cerebellar and cochlear granule cells [42], contrasts with the wide-spread expression of the (Y,, & and y2 subunits [21,25.47]. Expression of the clustered genes also appears to be separable during embryological development [ 13,261, in the staggerer neurological mutant [30] and after chronic drug treatment. For example, chronic ethanol treatment decreases mRNA lev-
The physioilogicml and phasmacologicaP ftinctiqns of the Q%BA,, G<, subunit are not well-tmdet.,stood. G/&A,4 receptors containing C+ subunits do not respond to benzodiarcpineh in vitro [X] and are claas~fied as ‘benzodiazepine-insensitive’ receptors. These receptors respond to several other classes of sedative hypnotics, including barbiturates and neurosteroids [ 171. ANT rats wlaicb display an enhanced sensitivity to benzodiazepines, ethanol and barbiturates [18]. have a point mutation in the (Ye subunit which confers diazepsm sensitivity to this normally insensitive subunit [23]. Chronic ethanol increases the levels of diazepam-insensitive receptors and the mRN.4 of the (Ye subunit in the cerebellar granule layer [33.48]; however, receptors containing the cy,, subunit expressed in vitro do not respond to ethanol [23]. Further research is needed to determine the functional roles of the GABA, receptor (Y,, subunit. The mouse is an animal species ideally suited for behavioral pharrnazogenetic studies using quantitative trait loci (QTL) analysis [6]. apping of the murine Gabm6 gene and identification of an RFLP for the fyh subunit in the mouse may provide a new means for probing the behavioral functions of the (Y,, subunit.
We would like to thank Dr. John Belknap for his discussions and assistance in editing this manuscript. This work was supported by grants from the Presbyterian Health Foundation (to K.M.G. and T.W.S.), the Nationa! Institutes of Mental HeaiKh (MH-l9791) (to T.W.S.) and the Narional Institute for Neurological Diseases and Stroke (NS I X089) (ta A.R.).
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to distal 5q by linkage analysis. Genotnks,
14 (1992)
745-748. I211 Kate. K.. Novel GABA +, receptor a subunit is expressed only in
cerebellar granule cells. J. Mol. Biol.. 2 14 (1990)
619-624.
1221 Keir. W.J., Kozak. C.A., Chakrabotti, A.. Dietrich. R.A. and Sikela,
J-M.. The cDNA
sequence and chromosomal localization
of the
mutine GABA, p 1 receptor gene. Genornks. 9 (199 I) 390-395. [23] KnolL J-K Sinnett. D.. Wagstaff. J.. Glatt, K., Wilcox, A.S.. Whiting. P.M.. Wingrove. P.. Sikela. J.M. and Laiande, M., FISH
ordering of reference markers and of the gene for the a5 subunit ot the y-aminobutyric acid receptor (GAERAS) within the Angelmann and Prader-Willi syndrome chromosomal regions. Num. Mol. Gerrct.. 2 (1993) 183-189. [24] Korpi, E.R.. Kleingoor. C.. Kettenmenn. H. and Seeburg. P.H.. Benzodiazepine-induced molar impaimlent linked to poinr muration in cerebellar GABA,, receptor. Ncfrffrc,. 361 (1993)356-3S). [2.5] Laurie, D.J., Wisden. W. and Seeburg, P.H.. Distribution of thirteen GABA, receptor subunit mRNAs in the rat brain. II. Olfactory bulb and cerebellum. J. Neurosci.. 12 (1992) lO63- 1076. [26] Laurie. D.J.. Wisden. W. and Seeburg. P.H.. Distribution of thirteen GABA, receptor subunit mRNAs in the rat brain. 111.Embryologic and postnatal development. J. Newnwi.. I2 (1992) 4 I5 I-4172. [27] Lin. L.-H., Whiting. P. and Harris. R.A., Molecular determinants of general anesthetic action: role of GABA, recepror structure. J. Nertrochem., 60 ( 1993) l548- 1553. [28] Lossie. A.C.. MacPhee. M.. Buchberg. A.M. and Camper. S.A.. Gmmre.5 ( 1994)SIt% I X0. Mouse chromosome 1I, Marw~c. (291 Luddens. H.. Pritchett. D.B.. Kohlcr. M.. Killisch. 1.. Keinanen. I<.. Monyer. H.. Sprengel. R. and Seeburg. P.H.. Cerrbellar GABA,\ receptor selective for a behavioral h!cohc,l antagonist, Nu~urlcrr.316 ( 1990) 648-65 I. [XI] Luntz-Lrybmnn. V.. Rotter. A.. Zdilar. D. and Frostholm. A.. Uncoupling of GABA, /benzodiazepine receptor alphal. beta2 and gamma2 subunit mRNA expression in cerrbellar purkinje cells ul Staggerer mutant mice, J. Neurosci.. 15 (1995)8 I2 I-8 130. 1311 Macdonald, R.L. and Olsen. R.W., GABA, receptor channels, Amur. Rer. Nelrrosci., 17 (1994) 569-602. [32] Manly, K.F., A Macintosh program for storage and analysis of experimental gene mapping data. Mun~rn. Grnonrr, 4 ( 1993) 303-3 I3 (MAP MANAGER 2.6.5 accessible at URL: HTTP://mcbio.med.buffalo.edu/mapmgr.html~. [33] McLean. P.J.. Farb. D.H. and Rusaek. S.J. Mapping of the LY, subunit gene (CABRA4) to human chromosome 4 defines an (Ye-(Y,PI-y, gene cluster: further evidence that modem GABA, receptor gene clusters are derived from an ancestral cluster. Gum~rt~ics. 26 (1995) 580-586. [34] Mhatrr. M. and Ticku. M.K.. Chronic ethanol administration incrcascs the binding ol the bsncodia~epine Inverse agonist and alcohol antagonist [’H]Rol S-45 I3 in rat brain. Eur. J. Plrcrrmcccwl.. IS3 (1988) 141-14s. [35] Nakatsu. Y.. Tynda!c. R.F.. DeLorey. T.M., Durham-Pierre, D.. Gardner, J.M.. McDaniel, H.J., Nguyen, Q., Wagstaff, J., Lalande. M.. Sikela, J.M.. Olsen. R.W., Tobin, A.J. and Brilliant. M.H., A cluster of three GABA,, receptor subunit genes is deleted in a neurological mutant of the mouse p locus. Nuture. 364 (1993) 448-450. [36] Olsen. R.W.. Drug interactions at the GABA receptor-ionophore complex. Annu. Rev. Phormucol. Toxicol.. 22 ( 1982) 245-277. 1371 Rowe. L.B., Nadeau. J.H., Turner, R., Franked. W.N.. Letts, V.A.. Eppig. J.‘T.. Ko, M.S.H.. Thurston, S.J. and Birkenmeier, E.H., Maps from two interspecific backcross DNA panels available as a community genetic mapping resource, Mumm. Gerwne. 5 (I 994) 253-274. [38] Russek. S.J. and Farb. D.H.. Mapping of the & subunit gene to microdissected human chromosome 5q34-q35 defines a gene cluster for the most abundant GABA,
receptor isoform, Gemmkx.
23
(1994) 538-533. 1391 Sambrook, J.. Fritsch. E.F. and Maniatis. T.. Molecular Ckmhg. A fuburatory Mw~ual. 2 edn., Cold Spring Harbor Laboratory Press. Cold Spring Harbor, NY, 1989. 1401 Silver. J.. Confidence limits for estimates of gene linkage based on analysis of recombinant inbred strains, Heredifi, 76 (1985) 436-440. 1411 Sommer. B.. Pousti&
A.. Spurr, N.K. and Secburg. P.H., The murine GABA, receptor &subunit gene: structure and assignment to h;lman chromosome I. DNA Cell Biol.. 9 (1990) 561-568. 1421 Varecka, L.. Wu. C.-H.. Rotter. A. and Frostholm. A.,
h, Jiangpillg
Gan
a7
Abstract We have established that the GABA ,, receptor q, (Grrhln6 1 and a, CGdmrl~ subunit penes are tightly linked on mouse chromosome I I by analyzing the strain distribution patterns of RFLPs for the two genes and microsatellite markers flanking these genes in 26 BXD recombinant
inbred strains. These results further demonstrate clustering of the GABA,,
receptor subunit penes on mouse chromosomes
and the synteny for these clusters between the mouse and human penomes. Kqxwz&t
GABA,,
receptor: Alphal zuhunil: Alpha6 subunit: RFLP; C57BLj6J
y-Aminobutyric acid (GABA) is the predominant inhibitory neurotransmitter ii; the mammalian central nerreceptors are the site of action vous system [36]. GABA, of several classes of therapeutically important drugs, including the benzodiazepines, barbiturates and inhalation anesthetics [27,36]. The GABA,, receptor is a heteropentamer composed of varying combinations of cr, ,8, y and S subunits. To date, 13 genetically distinct subunits have been identified in mammals, including 6 cy, 3 /3, 3 y and 1 6 species [3 I]. The electrophysiological and pharmacological properties of the GAIBA, receptor are dependent upon the subunit composition of the complex [31]. While strong evidence suggests that several isoforms of the GABA, receptor exist in vivo, the exact composition of individual GABA, receptor pentamers has yet to be determined. Many of the GABA, receptor subunit genes have been localized on human chromosomes. Most of these genes are grouped in cluPters. The CV:(GabruZ), (Y_,(Gd~rur’h PI (Gubrbl) and y, (Gczbrgl) genes are located on human chromosome 4 [4,8,33]. 4. second cluster, including the cyI (Gabrul), (Ye (Gabm6~, Pr (Grrbr172) and y2 (Gabrg2)
* Corresponding author. Department of Physiology. University of Oklahoma Health Sciences Center. PO Box 26901. Oklahoma City. OK 73190. USA. Fax: + I (405) 271-3181: E-mail: [email protected] 0169-328X/97/$17.00
and DBA/2J
genes, has been identified on human chromosome 5 [4,16,19,20.38,46]. The (Y~(Guhr-~.5), & (Gtrhrh3) and y3 (Ga1wg.Z) genes, located on human chromosome IS near the Angelmann-Prader Willi locus. comprise the third cluster [l-5,33,34]. Subunit genes not clustered with others include those for the LYE(G&Y& and 6 (G&Y!) subunits which are located on the human X chromosome and chromosome 1, respectively [2,41]. The mapped mouse GABA, receptor subunit genes are syntenic with those on the human chromosomes and are arranged in similar clusters. The Gabm2 and Gabrbl genes are located on mouse chromosome 5 [7] and the Gabrb3 and G&-g_? genes are tightly clustered on mouse chromosome 7 [7,35,43]. As in man, the Gabra3 gene is found on the mouse X chromosome [9]. Gabral and Gabrg2 genes are located on mouse chromosotne 11 [5,22]. The Gab&i gene has also been mapped to mouse chromosome 11 by interspecific backcross [37], however, its relationship to the (Y, and y, subunits was not determined. We have used restriction fragment length polymorphism (RFLP) and short sequence length polymorphism (SSLP) techniques in BXD recombinant inbred (RI) strains of mice to establish that the Gnbru6 gene is tightly linked to the G&-al and Gubrg2 genes on mouse chromosome 11. DNA from C57BL/6J and DBA/2J inbred mice and from 26 RI strains (obtained from Jackson Labs.) was
Copyright 0 1997 Elsevier Science B.V. All rights reserved.
P/ISO169-328X(96)00290-2
inbred strain: BXD recombinant inhred btrain
134
Hind III a, IBstE III a6 C57
DBA
C57
DBA
kb 23 ::t : 4.4 2.0 -
Fig. I. RFLPs for GABA, receptor (I, and q, subuni! probes in C57BL/6J and DBA/ZJ mice. Blots of C57BL/6J and DBA/?J genomic DNA digested with Hind111 were hybridized with a 1.8-kb cDNA probe of the human CABA, receptor (I , subunit. Blots of C57BL/6J and DBA/2J genomic DNA digested with BstEll were hybridized with a 264-bp cDNA probe of the mouse GABA, receptor a6 subunit.
isolated, digested with restriction enzymes, electrophoresed on 1% agarose gels and transferred to charged nylon membranes (Zeta Probe GT; Bio-Rad) [39]. The membranes were hybridized with cDNA clones for the (Y, or q, subunits random prime-labeled with [j2 P]dCTP (Promega). The probe used for the (Y, subunit was the full-length cDNA for the human (Y, subunit [14]. The
A
probe for the q, subunit was constructed by RT-PCR amplification of mRNA that encodes unique residues 322410 in the cytosolic loop between the TM3 and T regions of the C57BL/6J inbred mouse GABA, receptor LYEsubunit [21]. Genotyping of mouse microsatellites in inbred mice was performed by a modification of the method of Dietrich et al. [IO] as described by Bclknap et al. [1] using Mouse MapPair primers (Research Genetics). Products were electrophoresed on a native 8% polyacrylamide gel, stained with ethidium bromide and photographed. Linkage probability and the map position of the Gabru6 gene were determined from the strain distribution pattern of the RFLP and SSLP markers using the MAP MANAGER software [32]. A Hind111 RFLP for the LV, subunit was identified between progenitor C57BL/6J and DBA/2J inbred strains of mice (Fig. 1). Southern blots showed bands at 13. I, 2.6 and 1.2 kb in the C57BL/6J mice compared with 7.4, 5.3, 4.9, 2.8, 2.4 and 1.2 kb for the DBA/2J mice. We also observed a Nsil polymorphism in the LY,subunit between these two strains as predicted by Wang et al. [45] (data not shown). An RFLP also was identified for the ah subunit between the C57BL/6J and DBA/2J strains using BsrEII. A single band of 27.8 kb for the C57BL/6J and three bands of 19.6, 14.7 and 7.4 kb for the DBA/2J were seen on Southern blots (Fig. I). The strain distribution patterns of the (Y, and q, subunit RFLPs were characterized in the 26 BXD RI strains. The (Ye probe RFLP exhibited a distribution pattern among
BXD STRAINS 1
2
5
6
ii.8
P40-135
12.8
DllMit135
15.0
DllMit51
D D
17.1
DllMit174
Gabral
D D
la.2
DllMitZB
Ebf
25.0
I1 5 DllMi tZ3 DllMi t86
9 11 12 13 14 15 16 18 19 20 21 22 23 24 25 27 28 29 30 31 32 D D
DllMit53
D 0 D D R D
Gabra6
D D D D D 0 0
D
D D 0
Fig. 2. A: strain distribution pattern of Gabral and Gab&
D
in BXD recombinant inbred strains. B and D are generic symbols for alleles inherited from the
C57BL/6J and DBA/2J progenitors. respectively. Black (shaded) chromosomal regions representthose retaining markers from the C57BL/6J progenitor and white (unshaded) regions indic:lte those having alleles derived from the DBA/2J progenitor strain. A cross-over is indicated by break between the shaded and unshadedbars. using the MAP MANAGER
9:
partial linkage map of mouse chromosome I I was constructed from the data shown in A by minimizing cross-over events program version 2.6.5 1321.Genetic distancesare in CM. The map was anchored by assigning DI lMir2 to 5 CM [3].
RI maims i&mica8 with that Qf the a, subunjt RFLP (Fig. 2Al Using the data of EBlicPtt[12], we determined that t&c genes v ere Binked to the regionof nwia?ie ~~r(~~~~()~(~~~~e tp between IW-t-s.5 and 115 (Fit7 “&BP which is the same regio;a ?O wtlich GcrhwI had been previous8\:map@ by inter-subspecific backcross [5]. The G&r-0l and Gahrtraj geXS were flanked proximall) by DlldMir5l anJ di.siallj this Pinkage Sy by DIIMir20 and Ebf. We confirmed independently typing it51 and ~~~~~t2~. Strain distribution patterns for several other SSLP markers were characterized in the BXU R1 strains to identify markers tightly iinked to the GnBrul and G&r-lrh genes. One marker, DI lMitl74, was found to co-segregate with RFEPs for both GABA receptor genes. The assigned position of 51 IAJit13.5 I CM distal to P40-rsS and DIlilgir.53 [98] was confirmed by the observation of a single exchange: between these markers in line BXD-I (Fig. 2A.B). DllMit86 co-segregated with DilMir23 and 115 [I I]. Bur data establish that the murine Cdm~h gene is associated with the Gcrhrtrl-GdwgI cluster on mouse and G&rg-? chromosome I I. Bn man. the Gr,h/-~rl. Gtrhnh genes map to a restricted region on human chromosome 5q34-q35 [4,19.20,46]. These findings extend the synteny for this region conserved between mouse and man. The GABA, gene ercoding the ,8? subunit (G&&2) also has been mapped to the region of this cluster on human chromosome 5 [38]. The murine Cub162 gene has not yet been mapped, but is likely to be in the mouse chromosome 1i complex if synteny for this cluster of GABA, genes is maintained between man a;ld mouse. Our data from BXD RI analysis establish that the Gdwul and Gtrhw6 genes are likely to be within I CM (O-4 CM, 9.5% confidence limit) of one another (the limit of the resolution of these strains [40]). Wilcox et al. [46] showed that human Gahral and Gdwg.? are within 200 kb of one another. A precise c’
positioning of the human Ga61~6 gene relative to these two has not been reported. The functional significance of the clustering of these GABA, receptor subunit genes is unknown. One proposal is that clustered subunit genes may be coordinately regulated at the transcriptional level [46]. Additional molecular genetic data in both man and mouse is needed to determine
if the linkage of these genes is sufficiently close to allow co-regulation of their expression by common transcriptional regulatory sequences. The high degree of co-expression of transcripts for the lyi, & and -yz subunits among brain regions supports this idea [25,47]. However, other data suggest that independent regulation of these genes occurs. The restriction of cr6 transcription to only two regions of the mouse brain, the cerebellar and cochlear granule cells [42], contrasts with the wide-spread expression of the (Y,, & and y2 subunits [21,25.47]. Expression of the clustered genes also appears to be separable during embryological development [ 13,261, in the staggerer neurological mutant [30] and after chronic drug treatment. For example, chronic ethanol treatment decreases mRNA lev-
The physioilogicml and phasmacologicaP ftinctiqns of the Q%BA,, G<, subunit are not well-tmdet.,stood. G/&A,4 receptors containing C+ subunits do not respond to benzodiarcpineh in vitro [X] and are claas~fied as ‘benzodiazepine-insensitive’ receptors. These receptors respond to several other classes of sedative hypnotics, including barbiturates and neurosteroids [ 171. ANT rats wlaicb display an enhanced sensitivity to benzodiazepines, ethanol and barbiturates [18]. have a point mutation in the (Ye subunit which confers diazepsm sensitivity to this normally insensitive subunit [23]. Chronic ethanol increases the levels of diazepam-insensitive receptors and the mRN.4 of the (Ye subunit in the cerebellar granule layer [33.48]; however, receptors containing the cy,, subunit expressed in vitro do not respond to ethanol [23]. Further research is needed to determine the functional roles of the GABA, receptor (Y,, subunit. The mouse is an animal species ideally suited for behavioral pharrnazogenetic studies using quantitative trait loci (QTL) analysis [6]. apping of the murine Gabm6 gene and identification of an RFLP for the fyh subunit in the mouse may provide a new means for probing the behavioral functions of the (Y,, subunit.
We would like to thank Dr. John Belknap for his discussions and assistance in editing this manuscript. This work was supported by grants from the Presbyterian Health Foundation (to K.M.G. and T.W.S.), the Nationa! Institutes of Mental HeaiKh (MH-l9791) (to T.W.S.) and the Narional Institute for Neurological Diseases and Stroke (NS I X089) (ta A.R.).
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