Hippocampal cDNA Research Articles

Analysis of gene expression in the rat hippocampus using Real Time PCR reveals high inter-individual variation in mRNA expression levels.

In mammals, gene transcription is a step subjected to tight regulation mechanisms. In fact, changes in mRNA levels in the central nervous system (CNS) can account for numerous phenotypic differences in brain function. We performed a high-resolution analysis of mRNA expression levels for 37 genes selected from a normal rat hippocampus cDNA library. mRNA amounts were quantified using a Real Time PCR SYBR Green assay. We found that, in general, individuals from an inbred rat population (n = 20) have shown 2-3 times differences in the basal level of expression of the genes analyzed. Up to several fold differences among individuals were observed for certain genes. These inter-individual differences were obtained after correction for the different amounts of mRNA in each sample. Power calculations were performed to determine the number of individuals required to detect reliable differences in expression levels between a control and an experimental group. These data indicated that, depending on the variability of the candidate gene selected, it was necessary to analyze from five to 135 individuals in each group to detect differences of 50% in the levels of mRNA expression between two groups investigated. The comparison of mRNA abundance from different genes revealed a wide range of expression levels for the 37 genes, showing a 26,000-fold difference between the highest and lowest expressed gene.

Amida Predominantly Expressed and Developmentally Regulated in Rat Testis

Amida was first isolated from a rat hippocampal cDNA library as an Arc-associated protein. Previous studies showed that Amida is a nuclear protein and overexpression of Amida induces cell apoptosis. In this study, we found that Amida mRNA was expressed predominantly in rat testis by Northern blot analysis. During the development of testis, Amida mRNA was barely detectable until postnatal days 24 to 29 during which it increased to levels found in adults. However, Amida protein was not detected until postnatal day 32. Amida mRNA was found to be enriched in spermatocytes and less in round spermatids, but was undetectable in elongated spermatids by in situ hybridization. In addition, Amida protein was observed in the nucleus of spermatocytes and even in the elongated spermatids by immunohistochemistry. The development and cellular localization differences of Amida mRNA and protein implicates that Amida mRNA may undergo posttranscriptional regulation. Furthermore, Amida mRNA decreased significantly in the 8-day experimental cryptorchid testis when spermatogenesis was disrupted. Taken together, these data suggest that Amida is involved in spermatogenesis and may play an important role in development of testicular germ cells.

Cloning of a Novel G Protein-Coupled Receptor, SLT, a Subtype of the Melanin-Concentrating Hormone Receptor

A DNA fragment encoding an amino acid sequence possessing common features to the G protein-coupled receptor (GPCR) superfamily was found in the human genomic sequence, and from this information, the full-length cDNA of a novel GPCR, designated SLT, was cloned from the human hippocampus cDNA library. SLT showed the highest homology to the melanin-concentrating hormone (MCH) receptor, SLC-1 (31.5% identity), and to a lesser extent, to the somatostatin (SST) receptor subtypes. MCH exhibited agonistic behavior when applied to the SLT-expressing CHO cells at subnanomolar doses whereas more than 200 known peptides, including SST and cortistatin, did not. These results indicated that MCH is the cognate ligand of the SLT receptor and that this newly cloned GPCR is the second subtype of the MCH receptor. Quantitative polymerase chain reaction analysis of the SLT gene expression in human tissues showed that the SLT receptor is expressed mainly in brain areas including the cerebral cortex, amygdala, hippocampus, and corpus callosum, as well as in a limited number of peripheral tissues. The distribution of the SLT nearly overlapped that of SLC-1, suggesting that some of the neural functions of MCH may be mediated by both of these receptor subtypes.

Cloning and characterization of a novel variant of rat GABA BR1 with a truncated C-terminus

The γ-aminobutyric acid B receptor (GABA BR) belong to the G-protein-coupled receptor superfamily and has been identified as a mediator in the transmission of slow inhibitory neurotransmission in the mammalian central nervous system. Two types of GABA BR have been cloned, GABA BR1 and R2. GABA BR2 is co-expressed with GABA BR1 in many brain regions and inwardly rectifying potassium channels are activated by GABA BR agonists only upon co-expression of GABA BR1 with GABA BR2. Several splice variants of GABA BR1 receptors have been cloned from rat brain library. Using a rat hippocampal cDNA library, we have isolated a novel cDNA clone of GABA B receptor containing an insert of 124 bp between exon 3 and exon 4. This insert occurred between the regions encoding the Sushi domain and leucine binding protein (LBP)-like domain. The insert and subsequent frame shift generated a cDNA that codes for a truncated polypeptide of 239 amino acids lacking the C-terminus. Analysis of the deduced amino acid sequence of the new cDNA clone, termed GABA BR1g, showed that it was identical to the first 157 amino acids of GABA BR1a, but diverged thereafter. The C-terminal region of GABA BR1g contained two cysteine residues. GABA BR1g was expressed in both brain and peripheral tissues. Northern blot analysis demonstrated that two transcripts (4.5 kb and 4.0 kb) exist in hippocampus. In addition, studies of hippocampus in developing animals indicated that the expression of GABA BR1g is maximal at postnatal day four. GABA BR1g could be generated by alternative splicing of the GABA BR1 gene.

Cloning and tissue distribution of a novel isoform of the rat GABA(B)R1 receptor subunit.

We have identified a novel splice variant of the metabotropic GABA(B) receptor (R) subunit I, designated GABA(B)R1f, from a rat hippocampus cDNA library screen. GABA(B)R1f shares sequence homology with rat GABA(B)R1a, with the exception of an in-frame deletion of exon 4, resulting in a 21 bp deletion in the coding sequence of the N-terminal extracellular domain. In addition, GABA(B)R1f also contains a 93 bp in-frame insertion in a region of the sequence corresponding to the second extracellular loop and the fifth transmembrane domain, similar to that found in rat GABA(B)R1c. While being ubiquitously (but variably) expressed, reverse-transcription polymerase chain reaction analysis revealed the GABA(B)R1f isoform to be most prevalent in peripheral vs central tissues, suggesting a potential role for this novel isoform in either the mediation of inhibitory transmission in these various tissues, or in as yet defined actions unrelated to central synaptic regulatory mechanisms attributable to GABA(B)R.

Identification and characterization of PRKCBP1, a candidate RACK-like protein.

The receptors for activated C-kinase (RACK) family of proteins function as anchors for activated protein kinase C (PKC) isoenzymes. Using a monoclonal antibody to RACK1 in the screening of a human hippocampus cDNA library, we identified a novel member of the RACK family, designated PRKCBP1. Immunoprecipitation assays performed with a GST-fused PRKCBP1 protein suggest the carboxy terminus of PRKCBP1 interacts specifically with PKCbetaI. Northern analysis detected a 3.1-kb PRKCBP1 transcript in all tissues examined including brain, heart, liver, lung, pancreas, skeletal muscle, kidney, and spleen. The PRKCBP1 gene has been localized to human Chromosome (Chr) 20q12-13.1. Several groups have reported evidence for genetic linkage of Type II diabetes to this region in Caucasian families. This localization, combined with the observation that abnormalities in the activation, translocation, and inhibition of PKC occur in the development of Type II diabetes, suggested that PRKCBP1 was a candidate for contributing to Type II diabetes. We determined the PRKCBP1 coding sequence is 1845 bp in length, dispersed over 9 exons, spanning approximately 34 kb of genomic DNA. SSCP analysis was used to screen PRKCBP1 coding regions for mutations or polymorphisms in 100 Caucasian Type II diabetics and 100 Caucasian unaffected individuals. A silent C/T transition (bp1413, Thr137) was present in 23% of both diabetic and control individuals. A C/T transition (bp198) was also identified in a single diabetic individual, which resulted in no coding change (Ser66). The results of this analysis suggest that PRKCBP1 coding variations are uncommon and do not contribute to Type II diabetes in the general population.

Molecular cloning and expression of aminopeptidase A isoforms from rat hippocampus

The full-length cDNA encoding aminopeptidase A (APAL) was cloned from a rat hippocampus cDNA library. A short variant aminopeptidase A (APAS), produced by deletion, was also cloned. In the case of APAL, the longest open reading frame encodes 945 amino acid residues with a calculated molecular mass of 108 kDa, and the deduced amino acid sequence shows 76, 86 and 78% identity with its human, murine and porcine counterparts, respectively. Rat aminopeptidase A mRNAs were detected in the kidney, liver, heart and brain by Northern blot analysis. When overexpressed in COS-1 cells, APAL shows apparent aminopeptidase A activity, whereas APAS does not.

Molecular cloning of a novel member of the HSP110 family of genes, ischemia-responsive protein 94 kDa (irp94), expressed in rat brain after transient forebrain ischemia.

To identify genes induced by transient forebrain ischemia, we used the mRNA differential display technique in the four-vessel occlusion model in rats. Some genes were identified as candidates that encode ischemia-responsive protein, and one of them was cloned as ischemia-responsive protein 94 kDa (irp94) from the rat hippocampal cDNA library. Sequence analysis suggested that rat irp94 was a transcriptional variant or a homologue of mouse apg-2 and human heat shock protein (hsp) 70RY and a member of the HSP110 family, because IRP94 was >90% identical to APG-2 and HSP70RY and approximately 60% identical to the other members of the HSP110 family. Although irp94 mRNA was constitutively expressed in the normal hippocampus, it was clearly enhanced 4-24 h after ischemia for 10 (1.9-fold increase) and 15 min (3.4-fold increase). These changes mainly occurred in neuronal cells, as judged by the localization of irp94 mRNA using in situ hybridization histochemistry. On the other hand, hyperthermic stress did not enhance irp94 mRNA expression, suggesting that irp94 expression was enhanced under ischemic stress and not related to the heat shock signaling mechanism. Our study suggested that irp94, a novel member of the HSP110 family, might play an important role in the environment altering neuronal functions, especially after transient forebrain ischemia.

Kalirin Inhibition of Inducible Nitric-oxide Synthase

Nitric oxide (NO) acts as a neurotransmitter. However, excess NO produced from neuronal NO synthase (nNOS) or inducible NOS (iNOS) during inflammation of the central nervous system can be neurotoxic, disrupting neurotransmitter and hormone production and killing neurons. A screen of a hippocampal cDNA library showed that a unique region of the iNOS protein interacts with Kalirin, previously identified as an interactor with a secretory granule peptide biosynthetic enzyme. Kalirin associates with iNOS in vitro and in vivo and inhibits iNOS activity by preventing the formation of iNOS homodimers. Expression of exogenous Kalirin in pituitary cells dramatically reduces iNOS inhibition of ACTH secretion. Thus Kalirin may play a neuroprotective role during inflammation of the central nervous system by inhibiting iNOS activity.

Binding of Dynein Light Chain (PIN) to Neuronal Nitric Oxide Synthase in the Absence of Inhibition

PIN, an 89-amino-acid polypeptide found in a rat hippocampal cDNA library using the yeast two-hybrid system and various neuronal nitric oxide synthase (nNOS) fragments as bait, was reported to be an inhibitor of nNOS (Science274, 774–778, 1996). PIN reportedly inhibited nNOS selectively and did not interact with either the endothelial or inducible nitric oxide synthase isoforms. Inhibition was attributed to the ability of PIN to dissociate the catalytically active nNOS homodimer. PIN is a dynein light chain (J. Biol. Chem.271, 19358–19366, 1996), which suggested that PIN may serve as an axonal transport protein for nNOS. We have synthesized a rat PIN cDNA by recursive polymerase chain reaction and have expressed the protein inEscherichia coli.Recombinant PIN is a folded dimeric, mostly α-helical protein with a single deeply buried tryptophan residue. We have also expressed and purified the nNOS fragment to which PIN reportedly binds (residues 163–245). This recombinant peptide has a disordered secondary structure. Gel-filtration experiments show that PIN binds to both the full-length nNOS and nNOS fragment. However, PIN neither inhibits nNOS activity nor dissociates the nNOS dimer into monomeric species. PIN thus possibly functions as a dynein light chain involved in nNOS axonal transport but is not an inhibitor of the enzyme. Our results agree with the proposal (Cell82, 743–752, 1995) that the PIN recognition sequence in nNOS both lies outside the catalytic core and is not part of the monomer–monomer contact region.

Characterization of a partial cDNA clone detected by imidazoline receptor-selective antisera

A cDNA clone has been isolated from a human hippocampal cDNA expression library by relying on the selectivity of two antisera that are specific for imidazoline binding proteins. A 1789 bp cDNA clone was sequenced and shown to contain a single open-reading frame that predicts a 66 kDa polypeptide, but it is truncated based on its lack of a stop codon and poly-A + tail. Two regions of homology exist for the predicted amino acid sequence in common with chromogranin-A and B proteins, a zinc finger protein, and the ryanodine receptor. Northern blot analyses of poly-A + mRNA from 36 human tissues indicated two differentially expressed transcripts of 6.0 and 9.5 kb. The 6.0 kb mRNA form was enriched in brain and endocrine tissues as compared to other tissues, but not in strict concordance with I 1-imidazoline binding sites. The highest overall amounts of the combined transcripts were found in pituitary. In situ hybridization histochemistry revealed an enrichment of the message in neuronal cell bodies of the rat hippocampus and cerebellar cortex. This clone has some of the properties expected of an imidazoline receptor.

Cloning of a rat brain succinic semialdehyde reductase involved in the synthesis of the neuromodulator gamma-hydroxybutyrate.

The gamma-hydroxybutyrate biosynthetic enzyme succinic semialdehyde reductase (SSR) was purified to homogeneity from rat brain. Peptides were generated by tryptic cleavage and sequenced. PCR primers were designed from the amino acid sequences of two of the peptides showing a similarity (75-85%) to a mitochondrial aldehyde dehydrogenase. A PCR-amplified DNA fragment was generated from recombinant plasmids prepared by a mass excision procedure from a rat hippocampal cDNA library and used as a probe to screen this cDNA library. One cDNA of 1341 bp had an open reading frame encoding a protein of 447 residues with a deduced molecular mass of 47967 Da. The enzyme was expressed in Escherichia coli. Immunoblotting analysis revealed the existence of a protein with the same electrophoretic mobility as the SSR purified from rat brain and with an estimated molecular mass of 45 kDa. Northern blot experiments showed that this enzyme was not expressed in the kidney or in the liver. In the brain tissue, a single but rather broad band was labelled under high stringency conditions, suggesting the presence of more than one messenger species coding for SSR. Hybridization in situ performed on brain tissue slices showed specific labelling of the hippocampus, the upper cortex layer, the thalamus, the substantia nigra, the cerebellum, the pons medulla and the olfactory tract. The recombinant enzyme showed catalytic properties similar to those of the SSR purified from rat brain, particularly in regard to its substrate affinities and Ki for inhibition by phthalaldehydic acid. Valproic acid did not inhibit the cloned SSR. This enzyme had 20-35% identity in highly conserved regions involved in NADPH binding with four other proteins belonging to the aldo-oxo reductase family.

CDNA cloning and expression of a novel serine protease, TLSP

A cDNA for a putative novel serine protease, TLSP, was cloned from human hippocampus cDNA with polymerase chain reaction based strategies. The putative amino acid sequence of TLSP is similar to the trypsin-type serine proteases. TLSP mRNA is expressed in keratinocytes. Overexpressed TLSP protein in neuro2a cells was detected in culture medium.

Purification to Homogeneity and Properties of Plant Glucosidase I

Glucosidase I was purified about 3600-fold to apparent homogeneity from the microsomal fraction of mung bean seedlings. The purified enzyme removed the terminal α1,2-linked glucose from Glc3Man9GlcNAc2-peptide or the endoglucosaminidase H (Endo H)-released oligosaccharide. Glucosidase I activity was inhibited by kojibiose [Glc(α1-2)Glc], but not by other glucose disaccharides. Removal of up to four mannose residues from the N-linked oligosaccharide had little effect on its utilization as a substrate for glucosidase I. The enzyme had a subunit molecular weight of 97 kDa on SDS gels and this was shifted to 94 kDa after treatment with Endo H or Endo F, suggesting that glucosidase I is an N-glycoprotein having one oligomannose-type oligosaccharide. Amino acid sequences of this enzyme showed considerable identity to the enzyme cloned from a human hippocampus cDNA library. The enzyme was inhibited by castanospermine, deoxynojirimycin, MDL, and trehazolin, but not by australine or kifunensine. On the other hand, the other processing glucosidase, glucosidase II, is sensitive to inhibition by australine, but not by trehazolin. Thus, these two inhibitors are useful to distinguish glucosidase I from glucosidase II. The mung bean glucosidase I is quite sensitive to the histidine modifying reagent diethyl pyrocarbonate, whereas the pig liver glucosidase I is not. On the other hand, pig liver and pig brain glucosidase I preparations are sensitive to the sulfhydryl reagent NEM (N-ethylmaleimide), whereas the plant enzyme is not. These sensitivities to amino acid modifiers suggest significant differences between the plant and animal glucosidase I, in terms of catalytic site or protein conformation.

The Human Glycine Receptor β Subunit Gene (GLRB): Structure, Refined Chromosomal Localization, and Population Polymorphism

The glycine receptor of the human CNS comprises ligand-binding α1 and structural β subunits encoded by theGLRA1andGLRBgenes, respectively. Screening of a human hippocampal cDNA library resulted in the identification of the novel subunit transcript βB, differing in the 5′-UTR. Analysis of the genomic organization ofGLRBshowed that the coding region is distributed over nine exons, highly homologous to theGLRA1gene. Byin situhybridization, the chromosomal localization ofGLRBwas refined to band 4q31.3. Based on the identical phenotypes of mouse lines carrying mutant alleles of the α1 and β subunit genes,GLRBwas assumed to be a candidate gene for those cases of hyperekplexia that cannot be associated with mutations ofGLRA1.Therefore, flanking intronic sequences were determined, and DNA samples from more than 30 index patients were subjected to SSCP screening of the entireGLRBcoding region. A polymorphism in exon 8 was found both in the normal population and in families affected by hyperekplexia, although no coding mutation was detectable.

A novel orphan G protein-coupled receptor primarily expressed in the brain is localized on human chromosomal band 2q21.

A human hippocampus cDNA library was screened with a probe obtained from degenerate RT-PCR aimed at P2Y-homologous sequences. A positive clone, designated hip4, was identified containing an open reading frame of 1,020 bp that had been previously detected in a published genomic clone called R12. Subsequent screening of a human fetal brain cDNA library yielded a splice variant with a 1,104-bp open reading frame, which was named fb1. Both variants display the seven-transmembrane topology that is typical for G protein-coupled receptors. Probing a human multitissue northern blot revealed two brain-specific transcripts of 2.3 and 6.3 kb, respectively. Northern blot analyses with specific fragments confirmed that the two transcripts are generated by alternative polyadenylation yielding two different 3' untranslated regions. A genomic clone from the corresponding gene was isolated and mapped to human chromosomal band 2q21 by fluorescence in situ hybridization.

Molecular Cloning of a Peptidase Against N‐Acetylaspartylglutamate from a Rat Hippocampal cDNA Library

N-Acetylaspartylglutamate (NAAG) is the most prevalent peptide neurotransmitter in the mammalian nervous system. NAAG selectively activates the type 3 metabotropic glutamate receptor. It is inactivated by peptidase activity on the extracellular face of the plasma membrane of neurons and glia. The human gene that codes for prostate-specific membrane antigen (PSM) has been shown to produce peptidase activity against NAAG. We cloned the human PSM cDNA and used it to probe a rat hippocampal cDNA library. We identified a cDNA containing a complete coding region that possesses 83% homology with the PSM gene. The predicted 752-amino acid sequence has 85% identity and 91% similarity to the PSM sequence. CHO cells transfected with this cDNA expressed NAAG peptidase activity at a level similar to that obtained from rat brain membranes. The peptidase activity was inhibited by beta-NAAG, quisqualate, and pteroylglutamate but not aspartylglutamate or pteroic acid. In situ hybridization data demonstrated the widespread distribution of the peptidase mRNA in the brain, consistent with the distribution of peptidase activity. The highest levels of hybridization were detected in the hippocampus, dentate gyrus, piriform cortex, choroid plexus of the ventricles, pineal gland, anterior pituitary, and supraoptic nucleus. Three transcripts (estimated at 5, 3.4, and 2.9 kb) were identified in northern blots of rat brain, while in rat kidney the third transcript appeared slightly smaller than 2.9 kb. With use of reverse transcriptase PCR with primers for the 5' end, the central region, and the 3' end of the hippocampal cDNA, the expected amplification products were obtained from rat brain RNA. Spinal cord yielded an amplification product only with primers for the 5' end of the hippocampal cDNA.

Identification of an Efs isoform that lacks the SH3 domain and chromosomal mapping of human Efs.

Efs was originally found by expression cloning of a mouse embryo cDNA library through its Fyn-SH3 binding capacity (Ishino et al., Oncogene 11, 2331-2338, 1995). Efs has characteristic regions important in intracellular signal transduction; these are an SH3 domain, a cluster of putative ligands for SH2 domains and proline-rich sequences with SH3-binding consensus. In this paper, we report cDNA cloning of human Efs and a variant of it from a hippocampal cDNA library. The human Efs gene was mapped to chromosome 14q11.2-q12 by fluorescence in situ hybridization. We identified two forms of human Efs, designated hEfs1 and hEfs2. hEfs1 represents the human counterpart of original mouse embryo Efs (mEfs1). hEfs2, the newly identified form, is identical to hEfs1, except for its lack of the SH3 domain. hEfs1 and mEfs1 are 80% identical in their amino acid sequences and 100% identical within the SH3 domain. Reverse transcription polymerase chain reaction analysis of adult mouse tissue RNA indicated expression of Efs2 and of Efs1 in various tissues. Evidence suggesting the presence of the Efs2 protein in human tissue was obtained by immunoprecipitation followed by immunoblotting with two different anti-Efs antibodies. Possible functions of Efs2 are discussed.

Methylmalonyl-CoA Mutase Induction by Cerebral Ischemia and Neurotoxicity of the Mitochondrial Toxin Methylmalonic Acid

Differential screening of gerbil brain hippocampal cDNA libraries was used to search for genes expressed in ischemic, but not normal, brain. The methylmalonyl-CoA mutase (MCM) cDNA was highly expressed after ischemia and showed a 95% similarity to mouse and 91% similarity to the human MCM cDNAs. Transient global ischemia induced a fourfold increase in MCM mRNA on Northern blots from both hippocampus and whole forebrain. MCM protein exhibited a similar induction on Western blots of gerbil cerebral cortex 8 and 24 hr after ischemia. Treatment of primary brain astrocytes with either the branched-chain amino acid (BCAA) isoleucine or the BCAA metabolite, propionate, induced MCM mRNA fourfold. Increased concentrations of BCAAs and odd-chain fatty acids, both of which are metabolized to propionate, may contribute to inducing the MCM gene during ischemia. Methylmalonic acid, which is formed from the MCM substrate methylmalonyl-CoA and which inhibits succinate dehydrogenase (SDH), produced dose-related cell death when injected into the basal ganglia of adult rat brain. This neurotoxicity is similar to that of structurally related mitochondrial SDH inhibitors, malonate and 3-nitropropionic acid. Methylmalonic acid may contribute to neuronal injury in human conditions in which it accumulates, including MCM mutations and B12 deficiency. This study shows that methylmalonyl-CoA mutase is induced by several stresses, including ischemia, and would serve to decrease the accumulation of an endogenous cellular mitochondrial inhibitor and neurotoxin, methylmalonic acid.

The human glycine receptor β subunit: primary structure, functional characterisation and chromosomal localisation of the human and murine genes

The inhibitory glycine receptor (GlyR) is a pentameric receptor comprised of alpha and beta subunits, of which the beta subunit has not been characterised in humans. A 2106 bp cDNA, isolated from a human hippocampal cDNA library, contained an open reading frame of 497 amino acids which encodes the beta subunit of the human GlyR. The mature human GlyR beta polypeptide displays 99% amino acid identity with the rat GlyR beta subunit and 48% identity with the human GlyR alpha 1 subunit. Neither [3H]strychnine binding nor glycine-gated currents were detected when the human GlyR beta subunit cDNA was expressed in the human embryonic kidney 293 cell line. However, co-expression of the beta subunit cDNA with the alpha 1 subunit cDNA resulted in expression of functional GlyRs which showed a 4-fold reduction in the EC50 values when compared to alpha 1 homomeric GlyRs. Glycine-gated currents of alpha 1/beta GlyRs were 17-fold less sensitive than homomeric alpha 1 GlyRs to the antagonists picrotoxin, picrotoxinin and picrotin, providing clear evidence that heteromeric alpha 1/beta GlyRs were expressed. The beta subunit appears to play a structural rather than ligand binding role in GlyR function. Fluorescence in situ hybridisation was used to localise the gene encoding the human GlyR beta subunit (GLRB) to chromosome 4q32, a position syntenic with mouse chromosome 3. In situ hybridisation using the human GlyR beta subunit cDNA showed that the murine GlyR beta subunit gene (Glrb) maps to the spastic (spa) locus on mouse chromosome 3 at bands E3-F1. This is consistent with the recent finding that a mutation in the murine GlyR beta subunit causes the spa phenotype. It also raises the possibility that mutations in the human beta subunit gene may cause inherited disorders of the startle response.