Glutamic Acid Decarboxylase Gene Research Articles

Characterization of the glutamic acid decarboxylase gene promoter using transgenic mice

This article explores the actions of Chinese stakeholders as norm entrepreneurs in mobile Internet standard-making within the 3rd Generation Partnership Project (3GPP). Through semi-structured interviews with key experts from the Internet stakeholder communities, this article contextualises a rapidly transforming and increasingly politicised issue in the broader context of China's engagement with the global multistakeholder Internet governance architecture, as well as the debate on China's rise in the Liberal International Order. Furthermore, it incorporates the views and experiences of technologists working first-hand in standard-making, as they are often disregarded in political-scientific literature. Through the analytical lens of cognitivist regime theory, this article argues that the stronger China and Chinese stakeholders grow economically and politically, the more they become involved in the existing Internet governance regime complex, increasing their influence in the existing institutional arrangements without necessarily acting for changing their norms, rules, and principles. Through these theoretical and methodological approaches, new light is shed on the role of private and public Chinese stakeholders and on the relation between them.

209 Characterization of the glutamic acid decarboxylase gene promoter using transgenic mice

209 Characterization of the glutamic acid decarboxylase gene promoter using transgenic mice

Glutamic Acid Decarboxylase (GAD 67) Gene Expression in the Pancreas and Brain of the Nonobese Diabetic Mouse

A quantitative PCR was developed to measure GAD 67 mRNA levels in pancreas from NOD mice. Two nonautoimmune mouse strains were used as controls. An internal standard, rat GAD 67 mRNA, was prepared to quantify the reaction. In NOD mice GAD 67 expression in the pancreas was in the order of 2 × 106molecules/μg of total RNA; this expression was five times less in the two control mouse strains, whatever the age.The overexpression of GAD 67 characterized in the NOD mouse could be restricted to the pancreas since similar analysis performed in mouse brain did not reveal a large difference between the three mouse strains studied.

Glutamic acid decarboxylase-67 (GAD67): expression relative to GAD65 in human islets and mapping of autoantibody epitopes.

Glutamic acid decarboxylase (GAD), a target of both autoantibodies and autoreactive T-cells in insulin-dependent diabetes (IDD), exists as two homologous forms, GAD65 and GAD67. GAD65 is preferentially expressed in human islets and recognized by autoantibodies in IDD, but which form primarily elicits GAD autoimmunity is unknown. GAD67 gene expression in human islets has been demonstrated only by the polymerase chain reaction. We, therefore, quantitatively compared the expression of each GAD gene in human islets and mapped the binding of autoantibodies to recombinant human GAD67 by enzyme-linked immunosorbent assay. In ribonuclease protection assays, both forms of GAD messenger RNA (mRNA) were detected in human islets, although GAD65 mRNA was 200 times more abundant than GAD67 mRNA. Immunoblotting of islets with GAD form-specific antisera revealed GAD65, but not GAD67. By in situ hybridization and immunohistochemistry, GAD65 mRNA and protein were localized to islets, predominantly, but not entirely, to beta-cells; GAD67 mRNA and protein were undetectable. Thus, although GAD67 protein expression was undetectable in human islets, the GAD67 gene is transcribed, albeit weakly. Antibodies that recognized multiple epitopes in recombinant GAD67 were found in 20% of sera from ICA positive "at risk" first degree relatives of IDD subjects and recent-onset IDD subjects. The majority of GAD67 epitopes were mapped within the mid- and C-terminal thirds of the protein, a region that is highly conserved in GAD65. Although GAD67 may share cross-reactive epitopes with GAD65, these findings do not exclude the possibility that autoimmunity to GAD arises as a consequence of the aberrant up-regulation of GAD67 in human islets.

GABAergic modulation of striatal peptide expression in rats and the alterations induced by dopamine antagonist treatment

GABAergic modulation of enkephalin, substance P and glutamic acid decarboxylase (GAD67) gene expression and the alterations induced by dopamine receptor blockade were studied in the rat striatum. Following subchronic treatment with the GABA-A agonist muscimol, the GABA-B agonist baclofen or the GABA transaminase inhibitor γ-vinyl GABA there were no significant changes in striatal peptide and GAD67 gene expression. Following repeated administration of the D-2 antagonists, eticlopride and haloperidol, there was an increase in enkephalin and GAD67 mRNA levels and parallel decrease in that of substance P. These were unaffected by co-administration of γ-vinyl GABA. The D-1 antagonist, SCH 23390 administered alone or together with γ-vinyl GABA did not alter peptide or GAD67 mRNA levels. It seems that pharmacological stimulation of GABA receptors has little effect on enkephalin, substance P or GAD67 mRNA expression in striatal output neurons.

Localization and quantitation of expression of two glutamate decarboxylase genes in pancreatic beta-cells and other peripheral tissues of mouse and rat.

Glutamic acid decarboxylase (GAD) catalyzes synthesis of the inhibitory neurotransmitter gamma-amino butyric acid. Two homologous forms of GAD encoded by separate genes have been cloned from rat brain, with predicted protein sizes of 67 and 65 kilodaltons. GAD is present outside the brain, and pancreatic islet GAD is believed to be a target of autoimmunity in insulin-dependent diabetes mellitus. However, peripheral expression of the two GAD genes is incompletely characterized. We, therefore, investigated GAD expression in peripheral tissues, including pancreas, of mouse and rat. cDNAs encoding GAD 67 and GAD 65 were cloned from mouse brain and shown to be 95% homologous with the rat sequences. RNase protection assay using specific cRNA probes demonstrated expression of both GAD forms in freshly harvested pancreas and testis. Levels of both GAD mRNAs were greater in rat than mouse pancreas. GAD 67 mRNA was more abundant than GAD 65, and both were localized to islet beta-cells by in situ hybridization. In testis, both GAD mRNAs were localized to spermatocytes. Additionally, GAD 67, but not GAD 65, mRNA was detected in mouse and rat spleen and mouse liver. Thus, both GAD genes are expressed in peripheral tissues, with GAD 67 mRNA being more abundant under physiological conditions. The expression of both GAD 67 and GAD 65 genes specifically in islet beta-cells indicates that both GAD forms are candidate autoantigens in rodent models of insulin-dependent diabetes mellitus.

Localization and quantitation of expression of two glutamate decarboxylase genes in pancreatic beta-cells and other peripheral tissues of mouse and rat

Glutamic acid decarboxylase (GAD) catalyzes synthesis of the inhibitory neurotransmitter gamma-amino butyric acid. Two homologous forms of GAD encoded by separate genes have been cloned from rat brain, with predicted protein sizes of 67 and 65 kilodaltons. GAD is present outside the brain, and pancreatic islet GAD is believed to be a target of autoimmunity in insulin-dependent diabetes mellitus. However, peripheral expression of the two GAD genes is incompletely characterized. We, therefore, investigated GAD expression in peripheral tissues, including pancreas, of mouse and rat. cDNAs encoding GAD 67 and GAD 65 were cloned from mouse brain and shown to be 95% homologous with the rat sequences. RNase protection assay using specific cRNA probes demonstrated expression of both GAD forms in freshly harvested pancreas and testis. Levels of both GAD mRNAs were greater in rat than mouse pancreas. GAD 67 mRNA was more abundant than GAD 65, and both were localized to islet beta-cells by in situ hybridization. In testis, both GAD mRNAs were localized to spermatocytes. Additionally, GAD 67, but not GAD 65, mRNA was detected in mouse and rat spleen and mouse liver. Thus, both GAD genes are expressed in peripheral tissues, with GAD 67 mRNA being more abundant under physiological conditions. The expression of both GAD 67 and GAD 65 genes specifically in islet beta-cells indicates that both GAD forms are candidate autoantigens in rodent models of insulin-dependent diabetes mellitus.

Glutamic acid decarboxylase gene expression in thalamic reticular neurons transplanted as a cell suspension in the adult thalamus

The goal of the present study was to determine whether alterations in neuronal morphology and connections in thalamic grafts were accompanied by changes in the expression of mRNA encoding glutamic acid decarboxylase (GAD), the key enzyme in the synthesis of GABA, the normal neurotransmitter of neurons of the thalamic reticular nucleus. Cell suspensions of rat fetal tissue containing both thalamic reticular nucleus and ventrobasal primordia were transplanted into the excitotoxically lesioned somatosensory thalamus of adult rats. Levels of messenger RNA (mRNA) encoding GAD (Mr 67,000; GAD67) were measured 7 days to 4 months following transplantation via quantitative in situ hybridization with 35S-radiolabeled antisense RNAs. Expression of GAD67 mRNA in the thalamic reticular nucleus was analyzed in parallel in rat pups between 0 and 30 days postnatally, and in adult animals. As already observed with immunohistochemistry, transplanted neurons of the thalamic reticular nucleus did not group in specific clusters but rather mingled with unlabeled (putatively ventrobasal) neurons. Levels of labelling for GAD67 mRNA per neuron increased over time and reached adult levels during the third week post-grafting, i.e. 2 weeks after the theoretical birthdate of the neurons (grafted at embryonic days 15–16). Similar values were observed and a plateau was reached at similar time points during normal ontogeny. The results suggest that, in contrast to morphology and size of the neuronal cell bodies, gene expression of GAD67 develops normally despite the ectopic location of neurons of the thalamic reticular nucleus in the somatosensory thalamus, the abnormal connectivity and the lack of segregation from non-GABAergic neurons. Therefore, dissociation and transplantation of the tissue, resulting in disruption of the extracellular matrix and alteration of cellular interactions, differentially affects various aspects of neuronal maturation. Because neuronal activity in the graft is similar to that in the adult thalamic reticular nucleus, the results further suggest that, as shown in other experimental systems, GAD67 mRNA levels in the graft are regulated in relation to the functional activity of GABAergic neurons.

ESTABLISHMENT OF A STABLE GLUTAMATE DECARBOXYLELASE (GAD) EXPRESSING CELL-LINE BY TRANSFECTION.

We have constructed a recombinant DNA clone containing the gene encoding glutamic acid decarboxylase (GAD), which catalyzes the synthesis of γ-aminobutyric acid (GABA). This recombinant DNA was then transfected into mouse NIH-3T3 fibroblast cells for transplantation into Swiss-Web mice. In order to construct a plasmid capable of transcribing the DNA insert in the eucaryotic cells, the GAD gene was removed from pSP65-13, and was ligated into the vector pSV2neo, which contains the SV40 early promoter, and the neomycin resistance gene. The pSV2GAD was then transfected into NIH-3T3 fibroblasts by calcium phosphate precipitation, or by electroporation. The transfected fibroblasts were then selected with antibiotic G418 for amplification. The transient expression of GAD in the transfected fibroblasts was detected by immunocytochemical staining using anti-GAD antibody. A small population of GAD immunoreactive cells were clearly stained, and were easily distinguished from the majority of unstained background cells. These GAD-immunoreactive cells were not seen in either mock-transfected, or pSV2neo-transfected cells (vector-alone control). The transfected fibroblasts were continuously selected with antibiotic G418. Six out of 35 subcultures that had GAD-positive immunostaining in the cell lines were selected. Granular GAD-positive staining was observed in the cytoplasm and fiber extensions of the transfected cell lines in varying densities. The GAD-mRNA was also detected in the subcultures by in situ hybridization using a 35S-labeled 369-nucleotide riboprobe in pBluescript. The GAD-transfected NIH-3T3 cells were then transplanted into Swiss-Web mice. Fifteen to 30 days later, transplanted animals were perfused for identification. These cells were first identified with anti-fibronectin antibody, and the adjacent sections with anti-GAD or anti-GABA antibodies. All the transplants are fibronectin-positive. Both GAD- and GABA positive cells were observed in the transplant.

Many spinal cord cells transiently express low molecular weight forms of glutamic acid decarboxylase during embryonic development

At early developmental stages in the rat spinal cord (embryonic day 13), when neuronal progenitors are still proliferating, most differentiating neurons express truncated forms of glutamic acid decarboxylase (GAD) (approximately 25 kDa) which are the products of alternative splicing of the GAD 67 gene. These truncated proteins do not appear to synthesize γ-aminobutyric acid (GABA). The amino acid is detected in cells only after alternative splicing of the GAD 67 gene generates a full-length, 67 kDa enzymatically active form of GAD. Both the 67 kDa GAD and GABA colocalize and appear diffusely distributed in the cytoplasm of embryonic neurons. GABA does not appear associated with synaptic vesicles until after birth, when its intracellular distribution becomes punctate and it colocalizes with synaptophysin. At this time, it also colocalizes with an immunologically distinct 65 kDa GAD protein encoded by a second GAD gene (GAD 65). Expression of different GAD-related proteins with distinct intracellular distributions during development suggests that GABA, the product of these enzymes, may have trophic or metabolic roles during spinal cord differentiation.

Expression of the genes coding for glutamic acid decarboxylase in pluripotent cell lines

The expression of glutamic acid decarboxylase (GAD) is a basic characteristic of a wide array of inhibitory neurons that use γ-aminobutyric acid as a neurotransmitter. Clonal cell models will be essential for investigating the mechanisms which are responsible for the selective expression of GAD. P19 embryonal carcinoma cells are an important model for the analysis of neuronal gene expression. Depending on culture conditions, undifferentiated cells can be induced to form cells as widely divergent as cardiac muscle-like cells and neuron-like and glial-like cells. P19 cells are amenable to a number of powerful genetic manipulations including transformation with foreign DNA and selection of mutants. In this study we used nuclease protection assays and Northern blot analysis to determine if P19 cells express the GAD1 and GAD2 genes. The results show that uninduced P19 cells express these genes at very low but easily detectable levels. When the cells are induced to differentiate along the neuronal pathway with retinoic acid, the levels of transcripts for both GAD genes rise dramatically. At least some RNA transcripts of both genes from induced cells comigrate with the corresponding mRNA from the brain and thus probably represent processed mRNA. The expression of GAD genes in undifferentiated cultures of embryonal stem (ES) cells was also investigated. These cultures express levels of GAD1 transcripts that are higher than uninduced P19 cells. In contrast, expression of the GAD2 gene was barely detectable. These results indicate that P19 EC cells and ES cells will be useful for the investigation of the mechanisms that regulate the expression of the GAD1 and GAD2 genes.

Contrasting patterns in the localization of glutamic acid decarboxylase and Ca 2+ /calmodulin protein kinase gene expression in the rat centrat nervous system

The expression of the genes encoding the alpha subunit of type II calcium calmodulin-dependent protein kinase (CaM II kinase α) and the 67,000 mol. wt form of glutamic acid decarboxylase was examined throughout the rat centrat nervous system. In situ hybridization histochemistry, using cRNA probes, revealed a dense population of CaM II kinase α-expressing cells throughout the telencephalon and diencephalon. CaM II kinase α mRNA was also expressed in the midbrain, cerebellum and medulla oblongata, but at greatly reduced levels. No CaM II kinase α gene expression was detected in nuclei producing monoamines or acetylcholine. By contrast, the glutamic acid decarboxylase gene was moderately to highly expressed throughout the centrat nervous system. In severat regions there was a complementarity in the distributions of cells expressing the glutamic acid decarboxylase or CaM II kinase α genes. Cells in certain nuclei such as the thalamic reticular nucleus or globus pallidus showed glutamic acid decarboxylase gene expression only; others such as the majority of the dorsal thalamic nuclei showed CaM II kinase α gene expression only. Severat regions contained both glutamic acid decarboxylase and CaM II kinase α expressing cells. However, simultaneous immunostaining for both proteins revealed only two regions where CaM II kinase α and glutamic acid decarboxylase immunoreactivity were colocalized: the cerebellar Purkinje cells and the commissurat nucleus of the stria terminalis. The results imply that CaM II kinase α is primarily expressed in non-GABAergic neurons. In severat regions CaM II kinase α mRNA is concentrated in nuclei known to contain populations of neurons that use excitatory amino acid transmitters.

Differential effect of functional olfactory bulb deafferentation on tyrosine hydroxylase and glutamic acid decarboxylase messenger RNA levels in rodent juxtaglomerular neurons

Expression of the dopaminergic phenotype in olfactory bulb (OB) juxtaglomerular neurons (constituting a population of periglomerular and external tufted cells) is dependent upon functional innervation by peripheral olfactory receptors. Loss of functional input in rodents, by either peripheral deafferentation or deprivation of odorant access, results in a profound decrease in the expression of juxtaglomerular tyrosine hydroxylase (TH). We have examined the effects of such treatments on the expression of the neurotransmitter biosynthetic enzyme glutamic acid decarboxylase (GAD), which is colocalized with TH in the majority of TH-containing juxtaglomerular neurons. Following either chemically induced OB deafferentation in adult mice or unilateral odor deprivation in neonatal rats, steady-state OB GAD messenger RNA levels remained essentially unchanged as assessed by Northern blot analysis 20-40 days after treatment. These results were confirmed by in situ hybridization analysis, which demonstrated a profound loss of juxtaglomerular TH messenger RNA but no accompanying decrease in regionally colocalized GAD message. Since GAD is found in nearly all dopaminergic OB cells, the preservation of juxtaglomerular GAD message implies that olfactory receptor neurons exert a differential transneuronal regulation of TH and GAD gene transcription.

Short-term dopaminergic regulation of GABA release in dopamine deafferented caudate-putamen is not directly associated with glutamic acid decarboxylase gene expression

In vivo microdialysis and in situ hybridization were combined to study dopaminergic regulation of γ-amino butyric acid (GABA) neurons in rat caudate-putamen (CPu). Potassium-stimulated GABA release in CPu was elevated following a dopamine deafferentation. Local perfusion with exogenous dopamine (50 μM) for 3h via the microdialysis probe attenuated the potassium-stimulated increase in extracellular GABA in CPu. Expression of glutamic acid decarboxylase (GAD) mRNA was also increased in the dopamine deafferented CPu. However, local perfusion with dopamine had no significant attenuating effect on the increased GAD mRNA expression. These findings indicate that dopaminergic regulation of GABA neurons in the dopamine deafferented CPu includes both a short-term effect at the level of GABA release independent of changes in GAD mRNA expression and a long-term modulation at the level of GAD gene expression.

Transplants of fetal substantia nigra regulate glutamic acid decarboxylase gene expression in host striatal neurons

Lesions of the dopaminergic innervation to the striatum result in increased activity of glutamic acid decarboxylase (GAD) and increased GAD mRNA in striatal GABAergic neurons. Here we show that solid transplants of dopamine-containing fetal mesencephalic tissue placed adjacent to the striatum can completely reverse the elevation of GAD mRNA in the striatum of adult rats with complete lesions of the nigrostriatal dopamine projections. The ability of the fetal transplants to re-establish control over gene expression in host target neurons indicates that there is a significant transneuronal influence of the transplanted neurons. Furthermore, striatal GAD mRNA levels appear to be a good marker of the functional impact of dopamine-producing transplants.

Differential gene expression for glutamic acid decarboxylase and type II calcium-calmodulin-dependent protein kinase in basal ganglia, thalamus, and hypothalamus of the monkey

In situ hybridization histochemistry, using cRNA probes, revealed a complementarity in the distributions of cells in the basal ganglia, basal nucleus of Meynert, thalamus, hypothalamus, and rostral part of the midbrain that showed gene expression for glutamic acid decarboxylase (GAD) or the alpha-subunit of type II calcium-calmodulin-dependent protein kinase (CAM II kinase-alpha). Cells in certain nuclei such as the thalamic reticular nucleus, globus pallidus, and pars reticulata of the substantia nigra show GAD gene expression only; others in nuclei such as the basal nucleus of Meynert, medial mamillary nuclei, and ventromedial hypothalamic nuclei show CAM II kinase-alpha gene expression only. A few nuclei, for example, the pars compacta of the substantia nigra and the greater part of the subthalamic nucleus, display gene expression for neither GAD nor CAM II kinase-alpha. In other nuclei, notably those of the dorsal thalamus, and possibly in the striatum, GAD- and CAM II kinase-expressing cells appear to form two separate populations that, in most thalamic nuclei, together account for the total cell population. In situ hybridization reveals large amounts of CAM II kinase-alpha mRNA in the neuropil of most nuclei containing CAM II kinase-alpha-positive cells, suggesting its association with dendritic polyribosomes. The message may thus be translated at those sites, close to the synapses with which the protein is associated. The in situ hybridization results, coupled with those from immunocytochemical staining for CAM II kinase-alpha protein, indicate that CAM II kinase-alpha is commonly found in certain non-GABAergic afferent fiber systems but is not necessarily present in the postsynaptic cells on which they terminate. It appears to be absent from most GABAergic fiber systems but can be present in the cells on which they terminate. This suggests that the kinase may be differentially engaged in pre- and postsynaptic functions at certain synapses.

Developmentally regulated expression of an exon containing a stop codon in the gene for glutamic acid decarboxylase.

In the adult rat brain, the gene for glutamic acid decarboxylase (GAD; L-glutamate 1-carboxy-lyase, EC 4.1.1.15) is expressed predominantly as a 3.7-kilobase transcript. Earlier data showed that embryonic brain expresses an RNA transcript distinct from the adult form; however, the exact structure of this form was not elucidated. Here, transcripts expressed in the embryonic but not the adult brain were cloned and analyzed. These transcripts include an exon not expressed in the adult inserted into coding sequence. The embryonic exon contains a stop codon that is in-frame with the coding sequence. The exon is found in genomic DNA within the GAD gene where it is flanked by introns with conventional splice sites. On the basis of these structural data, we propose the hypothesis that, early in brain development, transcripts encoding a truncated form of GAD are expressed. The deduced protein cannot function as a decarboxylase because the stop codon in the embryonic exon occurs upstream of the binding site for pyridoxal phosphate, an essential cofactor. Thus, alternative splicing plays a crucial role in the pathway leading to the development of functional GABAergic neurons. The central nervous system-derived cell lines B65 and C6 express a mixture of the adult and embryonic forms of GAD mRNA. They therefore are useful clonal models of central nervous system cells in the early phases of differentiation.

Ovarian Steroid Regulation of Glutamic Acid Decarboxylase Gene Expression in Individual Hypothalamic Nuclei

Abstract The possibility that steroids exert their effects on luteinizing hormone (LH) and prolactin release and female sexual behaviour via altering gamma-aminobutyric acid (GABA) synthesis within the hypothalamus was investigated. Ovariectomized rats were treated with either oil, 5 mug oestradiol benzoate (OB) or 5 mug OB plus 0.5 mg progesterone (P) 48 h later; autopsy was carried out 54 h after the OB injection. At this time, both steroid treatments stimulated prolactin release and lordotic activity. OB alone exerted a negative feedback effect on LH release while OB plus P stimulated an LH surge. The brains of the rats in these three treatment groups were microdissected and glutamic acid decarboxylase messenger nbonucleic acid (GAD mRNA) was estimated in specific hypothalamic areas obtained from individual brains; these areas included the preoptic area (POA), ventromedial nucleus (VMN), anterior medial portion of the zona incerta (Zl) and the arcuatemedian eminence area (ARC/ME). GAD mRNA concentrations were assessed by the slot-blotting technique and expressed as a ratio of GAD mRNA: beta-actin mRNA. Both steroid treatments significantly reduced GAD mRNA in the ARC/ME and Zl and OB plus P also reduced the concentration in the POA, while OB alone had no effect in this area. Neither treatment affected GAD mRNA in the VMN. These results indicate that when steroids stimulate LH and prolactin release and female sexual behaviour, they reduce GABA activity probably by reducing GABA synthesis in the POA, Zl and ARC/ME. This suggests that GABA normally has an inhibitory influence on these parameters. GABA synthesis does not however, appear to be involved in the negative feedback effects of steroids on LH release as measured 54 h after OB treatment.

Sequences homologous to glutamic acid decarboxylase cDNA are present on mouse chromosomes 2 and 10

The chromosomal locations of mouse DNA sequences homologous to a feline cDNA clone encoding glutamic acid decarboxylase (GAD) were determined. Although cats and humans are thought to have only one gene for GAD, GAD cDNA sequences hybridize to two distinct chromosomal loci in the mouse, chromosomes 2 and 10. The chromosomal assignment of sequences homologous to GAD cDNA was determined by Southern hybridization analysis using DNA from mousehamster hybrid cells. Mouse genomic sequences homologous to GAD cDNA were isolated and used to determine that GAD is encoded by a locus on mouse chromosome 2 ( Gad-1) and that an apparent pseudogene locus is on chromosome 10 ( Gad-1ps). An interspecific backcross and recombinant inbred strain sets were used to map these two loci relative to other loci on their respective chromosomes. The Gad-1 locus is part of a conserved homology between mouse chromosome 2 and the long arm of human chromosome 2.

Linkage Analysis in a Family with Dominantly Inherited Torsion Dystonia: Exclusion of the Pro-Opiomelanocortin and Glutamic Acid Decarboxylase Genes and Other Chromosomal Regions Using DNA Polymorphisms

A search for the defective gene causing torsion dystonia has been carried out in a family manifesting an autosomal dominant mode of inheritance of this movement disorder. Complete neurologic examination and establishment of lymphoblast lines have been carried out for over 50 members. Linkage analysis, using cloned DNA sequences and restriction fragment length polymorphisms, was evaluated by the LOD score method with requisite assumptions for mode of inheritance, age-of-onset and incomplete gene penetrance. Genes for pro-opiomelanocortin and glutamic acid decarboxylase, which have been implicated in the etiology of the disease in rat models, were excluded as being responsible for the disease state in this family. Other regions of the genome were also excluded using DNA probes for other genes and random "unique" sequences.