A sequence-ready BAC contig of the GABAA receptor gene cluster Gabrg1-Gabra2-Gabrb1 on mouse chromosome 5

Abstract

The type-A receptors for the neurotransmitter GABA (gamma-aminobutyric acid) are ligand-gated chloride channels that mediate postsynaptic inhibition. The functional diversity of these receptors comes from the use of a large repertoire of subunits encoded by separate genes, as well as from differences in subunit composition of individual receptors. In mammals, a majority of GABA(A) receptor subunit genes are located in gene clusters that may be important for their regulated expression and function. We have established a high-resolution physical map of the cluster of genes encoding GABA(A) receptor subunits alpha2 (Gabra2), beta1 (Gabrb1), and gamma(1) (Gabrg1) on mouse chromosome 5. Rat cDNA probes and specific sequence probes for all three GABA(A) receptor subunit genes have been used to initiate the construction of a sequence-ready contig of bacterial artificial chromosomes (BACs) encompassing this cluster. In the process of contig construction clones from 129/Sv and C57BL/6J BAC libraries were isolated. The assembled 1.3-Mb contig, consisting of 45 BACs, gives five- to sixfold coverage over the gene cluster and provides an average resolution of one marker every 32 kb. A number of BAC insert ends were sequenced, generating 30 new sequence tag sites (STS) in addition to 6 Gabr gene-based and 3 expressed sequence tag (EST)-based markers. STSs from, and surrounding, the Gabrg1-Gabra2-Gabrb1 gene cluster were mapped in the T31 mouse radiation hybrid panel. The integration of the BAC contig with a map of loci ordered by radiation hybrid mapping suggested the most likely genomic orientation of this cluster on mouse chromosome 5: cen-D5Mit151-Gabrg1-Gabra2-Gabrb1-D5Mit58- tel. This established contig will serve as a template for genomic sequencing and for functional analysis of the GABA(A) gene cluster on mouse chromosome 5 and the corresponding region on human chromosome 4.

Figure 1

A BAC-based STS/EST-content map of the Gabr gene cluster on mouse chromosome 5 (oriented with centromeric end at left and telomeric end at right). The relative positions of mapped STSs and ESTs are indicated at top, including the corresponding loci names (D5Buc). The isolated BAC clones are shown as horizontal lines with circles along the lines indicating positive STS hits. The STSs were developed from BAC insert ends (black), known genes (red), ESTs (green), and an SSLP marker D5Mit305 (blue). When an STS corresponds to a clone insert end, a yellow circle is present at the end of the clone from which it was derived. BAC clones were isolated from the C57BL/6J library (black lines), from the 129/Sv library (blue lines), or from the Research Genetics CITB library (*). The size of each BAC as determined by PFGE analysis is indicated. C57BL/6J BAC clones selected to represent the minimal tiling path are indicated by black arrowheads. The map is displayed with equal spacing between STS/EST markers and the depicted clones together span a distance of ∼1300 kb. The orientation of the 5′- and 3′-Gabrg1 markers with respect to surrounding STSs on the contig could not be determined.

Figure 2

RH map of central mouse chromosome 5. The genetic map of mouse chromosome 5 with some selected loci and their position in cM (from the centromere) shown for orientation (Kozak and Stephenson 1998). Corresponding regions of homology in the human genome are denoted (http://www3.ncbi.nlm.nih.gov/Omim/Homology). The RH map of central mouse chromosome 5 from this work is shown in the enlarged area. Indicated are the positions of D5Mit markers and new D5Buc markers isolated from BAC clones with their specified insert end in brackets (Table 1). Pairwise lod scores and distances in centirays are indicated between neighboring loci.The RH mapping data (vector scores) have been deposited at the European Bioinformatics Institute (http://www.ebi.ac.uk/RHdb/index.html).