Spinocerebellar ataxia type-1 and spinobulbar muscular atrophy gene products interact with glyceraldehyde-3-phosphate dehydrogenase
Spinocerebellar ataxia type-1 and spinobulbar muscular atrophy gene products interact with glyceraldehyde-3-phosphate dehydrogenaseBeena Koshy, Toni Matilla, Eric N. Burright1, Diane E. Merry2, Kenneth H. Fischbeck2, Harry T. Orr1 and Huda Y. Zoghbi*
Departments of Pediatrics and Molecular and Human Genetics, and Howard Hughes Medical Institute, Baylor College of Medicine, Houston, Texas 77030, USA, 1Departments of Laboratory Medicine and Pathology, Biochemistry and Institute of Human Genetics, University of Minnesota, Minneapolis, Minnesota 55455, USA and 2Department of Neurology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA
Received May 2, 1996;Revised and Accepted June 24, 1996
Spinocerebellar ataxia type1 (SCA1) is one of several neurodegenerative disorders caused by expansions of translated CAG trinucleotide repeats which code for polyglutamine in the respective proteins. Most hypotheses about the molecular defect in these disorders suggest a gain of function, which may involve interactions with other proteins via the expanded polyglutamine tract. In this study we used ataxin-1, the SCA1 gene product, as a bait in the yeast two-hybrid system and identified the glycolytic enzyme glyceraldehyde- 3-phosphate dehydrogenase as an ataxin-1 interacting protein. In addition, the yeast two hybrid data demonstrate that wild type and mutant ataxin-1 form homo and heterodimers. Physical interaction between GAPDH and ataxin-1 was also demonstrated in vitro. To investigate if GAPDH might interact with other glutamine repeat-containing proteins involved in neurodegenerative disorders, we tested its binding to the androgen receptor which is mutated in spinobulbar muscular atrophy. The androgen receptor interacts with GAPDH both in the yeast two-hybrid system and in vitro. The binding of both ataxin-1 and the androgen receptor to GAPDH does not vary with the length of the polyglutamine tract. While provocative, these findings do not address the selective neuronal loss in each of these disorders in light of the wide expression patterns of GAPDH and the respective polyglutamine containing proteins. Nonetheless, such interactions may increase the susceptibility of specific neurons to a variety of insults and initiate degeneration.
To date, five neurodegenerative disorders have been shown to be caused by expansions of CAG repeats which lie within the coding regions of their respective genes. These include spinobulbar muscular atrophy (SBMA), Huntington disease (HD), spinocerebellar ataxia type-1 (SCA1), dentatorubropallidoluysian atrophy (DRPLA), and Machado-Joseph disease (MJD) (1 ). The CAG repeat codes for a glutamine tract in each of the involved proteins and the length of the tract correlates inversely with the age of onset and severity of disease (2 ,3 ). In SCA1, normal alleles have a repeat number ranging from 6-44 usually interrupted with one or more CAT triplets when the CAG tract is longer than 21 repeats, while the SCA1 alleles have an unstable uninterrupted CAG repeat which ranges from 40-82 (4 ,5 ). The proteins implicated in each of these diseases are dissimilar except for the polymorphic polyglutamine tracts, and for most the normal function is unknown (2 ). In all five disorders, the transcripts for the respective genes are widely expressed while the neuro- degeneration is cell-specific. The molecular mechanism by which an expanded polyglutamine tract leads to cell-specific neuro- degeneration remains unknown.
Numerous observations argue against a loss of function mechanism in the pathogenesis of CAG-mediated neurodegenerative diseases. These include the absence of a HD-like phenotype in heterozygous individuals with deletions of the HD gene and the lack of motor neuron disease in males with deletions of the androgen receptor (AR), the gene implicated in SBMA (6 -8 ). Furthermore, gene targeting of the HD gene in mice leads to embryonic lethality in null mutant mice and apparently normal or subtle neurobehavioral phenotype in heterozygotes with no evidence of HD neuropathology (9 -11 ). The finding that in each of the mutated genes, the CAG repeats are found in the coding region and in each case encode a polyglutamine tract, suggests that this mutation exerts its effects at the protein level. Furthermore, transgenic mice carrying a human SCA1 cDNA with an expanded number of CAG repeats under the control of a Purkinje cell specific promoter develop ataxia and Purkinje cell degeneration (12 ). In SCA1 transgenic mice, the mutant SCA1 protein was demonstrated to be translatable and although it was not detected by western analysis, it was found at high levels in Purkinje cells by immunohistochemistry (Zoghbi and Orr, unpublished data). These data support the hypothesis that a gain of novel properties by the product of the expanded allele is responsible for the pathogenesis, at least in SCA1. The gain of function may be mediated by specific protein-protein interactions involving the expanded glutamine repeat and possibly a related set of molecules, which thus far remain unidentified.
To identify proteins that interact with the SCA1 gene product, we used the yeast two-hybrid system of interaction cloning (13 ,14 ). In this study, we report the identification of a glycolytic enzyme, the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an ataxin-1 interacting protein. Furthermore, we demonstrate that GAPDH also interacts with the androgen receptor, the gene product mutated in SBMA. The interaction of both ataxin-1 and the androgen receptor with GAPDH suggests that in SCA1 and SBMA, the onset of neuronal cell death may share common biochemical mechanisms and may in part be mediated through interaction with a common set of cellular proteins.
For the yeast two-hybrid screen, we used a fusion protein bait consisting of the yeast Gal4 DNA-binding domain and the ataxin-1 protein (amino acids 1-819) expressed from a yeast shuttle vector pAS2-CYH2 (see Materials and Methods). This bait plasmid was co-transformed into the yeast strain Y190 with a mouse embryonic cDNA expression library (10.5 days post coitum) fused to the Gal4 activation domain in the vector pACT2 (15 ). The mouse embryonic library was selected for identifying ataxin-1 interacting proteins because it is a high quality library and at this time there are no high quality human cerebellar libraries from either fetal or adult tissue available in the appropriate vectors for the two hybrid screen. Furthermore, we rationalized that there is no reason to assume a priori that the putative interactor will be expressed only in adult tissues. About 105 transformants were screened for activation of the HIS3 and LacZ ([beta]-galactosidase) reporter genes. After the isolation of Trp+, Leu+, His+, LacZ+ colonies, a chemical selection on cycloheximide media resulted in a strain that is devoid of the bait plasmid but carries the library plasmid (15 ). This assay eliminates putative interacting proteins which activate the reporter genes in the absence of the bait, ataxin-1. One of the positive clones, clone II.1 was identified as the glyceraldehyde-3-phosphate dehydrogenase gene (amino acids 1-170) by sequence analysis. To determine the specificity of the interaction, control experiments with dihybrid crosses showed that GAPDH specifically interacted with ataxin-1; matings of yeast containing the GAPDH clone with those expressing a battery of heterologous bait fusion proteins formed diploids that showed no detectable [beta]-galactosidase activity. In contrast, the Gal4-ataxin-1 strain exhibited strong [beta]-galactosidase activity when mated to yeast carrying either the library GAPDH clone (Fig. 1 ) or the full length rat GAPDH fused to the Gal4 activation domain in pGAD424 (data not shown). These experiments indicate that the interaction produced by these clones in the two-hybrid system is specific for ataxin-1 and is not due to the promiscuous interaction of GAPDH with random cellular proteins. Using the two-hybrid system we were also able to show that ataxin-1 self-associates (Fig. 1 ) and that the association occurs between wild type molecules or mutant molecules (homodimerization) as well as between wild type and mutant gene products (heterodimerization).
To delineate the region of the ataxin-1 protein that interacts with GAPDH, a set of constructs containing N-terminal and C-terminal deletions were created using the polymerase chain reaction, cloned into the pAS2-CYH2 plasmid and used to co-transform the HF7c strain which expresses the GAPDH clone. The pattern of interaction of ataxin-1 deletion mutants with GAPDH is summarized in Figure 2 . Ataxin-1 was found to interact with GAPDH through the N-terminal domain containing the polyglutamine stretch. In addition, clone Pre-N which contains the N-terminal portion of the protein minus the glutamine repeats showed binding to GAPDH albeit at a lower level than the clone N-N', which also contains the glutamine repeat region. This suggests that there is some specificity of binding of GAPDH to ataxin-1. None of the clones which contained only the carboxy terminal regions of ataxin-1 showed interaction with the enzyme. These results identify the N-terminal portion (amino acids 1-300) of ataxin-1 as critical for interaction with GAPDH.
To confirm the interaction observed in yeast, we examined whether GAPDH and ataxin-1 associate in vitro. Rabbit muscle GAPDH was immobilized on Affigel and incubated with either wild type ataxin-1 (32 repeats) or mutant ataxin-1 (82 repeats) expressed in COS7 cells. Following extensive washing, bound proteins were run on an SDS-polyacrylamide gel, blotted and probed with an anti-ataxin-1 antibody. As seen in Figure 3 a, both the wild type and mutant ataxin-1 proteins were specifically retained on the GAPDH beads. As a control, we used Affigel beads whose active groups had been blocked. No retention of either forms of ataxin-1 was observed using the beads alone (Fig. 3 b). The strength of the binding interaction was assessed using a gradient of NaCl washes. The binding of ataxin-1 to GAPDH persisted up to a 1 M NaCl wash (Fig. 4 ). Similar results were obtained when using COS7 cells expressing mutant ataxin-1 (data not shown). Elution of ataxin-1 from the GAPDH resin was only possible with 2% Formic acid (data not shown).
To determine if binding of ataxin-1 to GAPDH was unique or if it was a common feature of other glutamine repeat containing proteins involved in neurodegenerative disorders, we investigated the binding of the androgen receptor (AR) implicated in SBMA to GAPDH. First, we used the yeast two-hybrid system to demonstrate AR-GAPDH interaction. Amino terminal portions of the androgen receptor containing 24 (amino acids 13-134), 45 (amino acids 13-155) and 66 (amino acids 13-176) glutamines were cloned in pAS2 (Clontech) and transformed into the yeast strain Y187 for mating analysis. This yeast strain was then mated to HF7c which carries the GAPDH clone (amino acids 1-170 fused to the Gal4 DNA activation domain) or pGAD424 which carries the full length rat GAPDH cDNA. The resulting diploids were assayed for [beta]-galactosidase activity. Figure 6 a, shows that all three alleles of the androgen receptor interact with GAPDH. To confirm this interaction, full length androgen receptor constructs with 24, 45 and 66 CAG repeats were expressed in COS7 cells and protein extracts from these cells were incubated with GAPDH linked to the Affigel resin. Figure 6 b shows that all three androgen receptor proteins with varying repeat lengths are retained on the GAPDH resin. These results demonstrate that binding to GAPDH is not a phenomenon that is restricted to ataxin-1 but possibly is a feature of the proteins involved in other glutamine repeat disorders.
The plasmid vector pAS2-CYH2 used to construct the bait proteins has already been described (15 ). Full length wild type ataxin-1 cDNA with 30 repeats (pAS2-N4) or mutant containing 82 repeats (pAS2-E3) was cloned in frame in pAS2-CYH2 and their integrity confirmed by DNA sequencing. Similarly, cDNA coding for the N-terminal portions of the androgen receptor with 24, 45 and 66 repeats were cloned in the pAS2 vector (Clontech). Full length rat GAPDH was cloned in frame in the pGAD424 vector which contains the Gal4 activation domain. Yeast strains Y187, Y190 (15 ) and HF7c (37 ) have already been described.
Yeast strain Y190 was transformed with the bait plasmid pAS2-N4 and selected as previously described (14 ,38 ). The mouse embryonic library (10.5 d.p.c) tagged with the Gal4 activation domains in the pACT vector was obtained from Dr E. N. Olson. Y910 carrying the bait vector was transformed with the mouse embryonic library essentially as described (14 ). His+ colonies that were obtained were patched onto fresh plates and retested for the His+ phenotype and for [beta]-galactosidase activity using the filter-lift method (39 ). Library plasmids from His+, [beta]-gal+ colonies were recovered after growth of yeast on synthetic complete media plates lacking leucine (SC-Leu) plus cycloheximide 2.5 [mu]g/ml (15 ). Bait plasmid loss was confirmed by a Trp- phenotype before plasmid DNA was recovered for transformation of E. coli. DNA sequences were determined directly from the pACT2 plasmid containing the cDNAs by automated sequencing. To assess the specificity of the interaction of ataxin-1 with GAPDH, Y187 carrying either the partial (amino acids 1-170 in pACT2) or full length GAPDH in pGAD424 was mated (MAT[alpha] X MATa) with yeast strain Y190 carrying pAS2 plasmids expressing ataxin-1 (30 or 82 repeats) or heterologous bait proteins, lamin, SNF1, p53, cyclin D1, cdc 25, tat, cdk2, cdk6 to form diploids. In the diploids the association of expressed proteins results in the expression of the [beta]-galactosidase reporter gene which was assayed using the filter lift method.
COS7 cell extracts containing the wild type and mutant ataxin-1 proteins were prepared as previously described (12 ). Likewise, full length cDNAs for androgen receptor alleles containing 24, 45 and 66 repeats were cloned in pCMV5 vector, expressed in COS7 cells and cell extracts prepared (40 ). Extracts from lymphoblastoid cells from controls and SCA1 patients were prepared as described (3 ). In vitro binding assays were carried out as follows: Rabbit muscle glyceraldehyde-3-phosphate dehydrogenase (Sigma) was coupled to Affigel-10 (BIO-RAD) according to the manufacturers instructions. As a control, Affigel-10 treated with triethanolamine to block reactive groups was used. Approximately 50 [mu]g of total protein from COS7 cell extracts expressing either ataxin-1 alleles 30 or 82 repeats or the androgen receptor alleles 24, 45, and 66 glutamine repeats was used. In the case of the lymphoblastoid cell extracts from controls or SCA1 patients, 150 [mu]g of total protein was used. Protein extracts were diluted into 300 [mu]l of binding buffer [50 mM, HEPES (pH 7.4), 150 mM NaCl, 1 mM EGTA, 2 mM MgCl2, 1 mM PMSF, and 1 [mu]g/ml each of leupeptin, pepstatin and aprotinin] and incubated with 250 [mu]l of a 50% slurry of GAPDH coupled matrix for 1 h either at room temperature or 4oC. Following incubation, the resin was pelleted by centrifugation and the flow-through collected. The resin was then subjected to a series of washes, twice with 1 ml of binding buffer containing 0.05% Tween-20, followed by one wash each with 1 ml of 300 mM, 500 mM, and 1 M NaCl in binding buffer. After the last wash, the resin was centrifuged down and suspended in loading buffer and run on 10% SDS-PAGE gels. Proteins were detected by western blot analysis using antibody 11750V to ataxin-1 (3 ) and a mouse mAb F39.4.1 to the androgen receptor (41 ).
For quantitation of the strength of the interactions, yeast strains Y190 or HF7c transformed with the plasmids under study were grown to stationary phase in synthetic medium lacking leucine and tryptophan. Cultures were diluted 10-fold and regrown to a final A600 of 0.8-1.2. [beta]-galactosidase activity was determined as follows: 0.2 ml of yeast transformant extracts was added to 0.8 ml of Z buffer (60 mM Na2HPO4/40 mM NaH2PO4/10 mM KCl/1 mM MgSO4, 40 mM 2-mercaptoethanol, pH 7.0) and warmed to 28oC. Reactions were initiated by addition of 0.2 ml of o-nitrophenyl-[beta]-D-galactopyranoside (4 mg/ml) in Z buffer and terminated using 0.5 ml of 1 M Na2CO3 and A420 monitored. Values reported are the average of duplicate assays of five independent transformants. Specific [beta]-galactosidase activity is expressed as nanomoles per minute per milligram protein (42 ). To determine if the size of the CAG repeat significantly affects the ataxin-1-GAPDH interaction, Levene's test, a homogeneity-of-variance test was employed. The non-parametric Kruskal-Wallis one-way ANOVA was used to evaluate the GAPDH-AR interaction. All values are represented as mean +- standard deviation.
We thank Drs S. Elledge and W.J. Harper for vectors, yeast strains and control bait plasmids; E.N. Olson and Q. Ling for the mouse embryonic cDNA library; D. Beach for the HF7c yeast strain; E.M. Wilson for the AR COS7 constructs and A. Brinkmann for the mAb to the AR. B.K. thanks M. Edwards, A. McCall and M. Bhat for helpful discussions. This work was supported by grants from the National Institutes of Health Grants (NS27699) to H.Y.Z., (NS22920) to H.T.O., (NS32214) to K.H.F. and (NS09724) to E.N.B. and from the Muscular Dystrophy Association to K.H.F. and D.E.M. H.Y.Z. is an investigator with the Howard Hughes Medical Institute. T. Matilla is supported by the Spanish Ministerio de Educación y Ciencia (PF94 968798). Portions of this work were supported by core facilities of the Mental Retardation Research Center and the Child Health Research Center at Baylor College of Medicine.
2 Housman, D. (1995) Gain of glutamines, gain of function? Nature Genet., 10, 3-4, news & views.MEDLINE Abstract
3 Servadio, A., Koshy, B., Armstrong, D., Antalfty, B., Orr, H.T. and Zoghbi, H.Y. (1995) Expression analysis of the ataxin-1 protein in tissues from normal and spinocerebellar ataxia type 1 individuals. Nature Genet., 10, 94-98.MEDLINE Abstract
4 Chung, M.-Y., Ranum, L.P.W., Duvick, L.A., Servadio, A., Zoghbi, H.Y. and Orr, H.T. (1993) Evidence for a possible mechanism predisposing to intergenerational CAG repeat instability in spinocerebellar ataxia type 1. Nature Genet., 5, 254-258.MEDLINE Abstract
5 Quan, F., Janas, J. and Popovich, B.W. (1995) A novel CAG repeat configuration in the SCA1 gene: implications for the molecular diagnostics of spinocerebellar ataxia type 1. Hum. Mol. Genet., 4, 2411-2413.MEDLINE Abstract
6 Gusella, J.F., Tanzi, R.E., Bader, P.I., Phelan, M.C., Stevenson, R., Hayden, M.R., Hofman, K.J., Faryniarz, A.G. and Gibbons, K. (1985) Deletion of Huntington's disease-linked G8 (D4S10) locus in Wolf-Hirschhorn syndrome. Nature, 318, 75-78.MEDLINE Abstract
7 Quigley, C.A., Friedman, K.J., Johnson, A., Lafreniere, R.G., Silverman, L.M., Lubahn, D.B., Brown, T.R., Wilson, E.M., Willard, H.F. and French, F.S. (1992) Complete deletion of the androgen receptor gene: definition of the null phenotype of the androgen insensitivity syndrome and determination of carrier status. J. Clin. Endocrinol. Metab., 74, 927-933.MEDLINE Abstract
8 Trifiro, M., Gottlieb, B., Pinsky, L., Kaufman, M., Prior, L., Belsham, D.D., Wrogemann, K., Brown, C.J., Willard, H.F., Trapman, J., Brinkmann, A.O., Chang, C., Liao, S., Sergovich, F. and Jung, J. (1991) The 56/58 kDa androgen-binding protein in male genital skin fibroblasts with a deleted androgen receptor gene. Mol. Cell. Endocrinol., 75, 37-47.MEDLINE Abstract
9 Nasir, J., Floresco, S.B., O'Kusky, J.R., Diewert, V.M., Richman, J.M., Zeisler, J., Borowski, A., Marth, J.D., Phillips, A.G. and Hayden, M.R. (1995) Targeted disruption of the Huntington's disease gene results in embryonic lethality and behavioral and morphological changes in heterozygotes. Cell, 81, 811-823.MEDLINE Abstract
10 Zeitlin, S., Liu, J.-P., Chapman, D.L., Papaioannou, V.E. and Efstratiadis, A. (1995) Increased apoptosis and early embryonic lethality in mice nullizygous for the Huntington's disease gene homologue. Nature Genet., 11, 155-163.MEDLINE Abstract
11 Duyao, M.P., Auerbach, A.B., Ryan, A., Persichetti, F., Barnes, G.T., McNeil, S.M., Ge, P., Vonsattel, J.-P., Gusella, J.F., Joyner, A.L. and MacDonald, M.E. (1995) Inactivation of the mouse Huntington's disease gene homolog Hdh. Nature, 269, 407-410.
12 Burright, E.N., Clark, H.B., Servadio, A., Matilla, T., Feddersen, R.M., Yunis, W.S., Duvick, L.A., Zoghbi, H.Y. and Orr, H.T. (1995) SCA1 transgenic mice: a model for neurodegeneration caused by an expanded CAG trinucleotide repeat. Cell, 82, 937-948.MEDLINE Abstract
13 Fields, S. and Song, O.-K. (1989) A novel genetic system to detect protein-protein interactions. Nature, 340, 245-246.MEDLINE Abstract
14 Durfee, T., Becherer, K., Chen, P.-L., Yeh, S.-H., Yang, Y., Kilburn, A.E., Lee, W.-H. and Elledge, S.J. (1993) The retinoblastoma protein associates with the protein phosphatase type 1 catalytic subunit. Genes Dev., 7, 555-569.MEDLINE Abstract
15 Harper, J.W., Adami, G.R., Wei, N., Keyomarsi, K. and Elledge, S.J. (1993) The p21 Cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinases. Cell, 75, 805-816.MEDLINE Abstract
16 Ercolani, L., Florence, B., Denaro, M. and Alexander, M. (1988) Isolation and complete sequence of a functional human glyceraldehyde-3-phosphate dehydrogenase gene. J. Biol. Chem., 263, 15335-15341.MEDLINE Abstract
17 Harris, J.I. and Waters, M. (1976) In Boyer, P.D. (ed.), The Enzymes. Academic Press, Boca Raton, Vol. 13, pp. 1-49.
18 Brodie, A.E. and Reed, D.J. (1987) Reversible oxidation of glyceraldehyde 3 phosphate dehydrogenase thiols in human lung carcinoma cells by hydrogen peroxide. Biochem. Biophys. Res. Comm., 148, 120-125.MEDLINE Abstract
19 Burke, J.R., Enghild, J.J., Martin, M.E., Jou, Y.-S., Myers, R.M., Roses, A.D., Vance, J.M. and Strittmatter, W.J. (1996) Huntingtin and DRPLA proteins selectively interact with the enzyme GAPDH. Nature Med., 2, 347-350.
20 Fox, J.B. and Dandliker, W.B. (1956) A study of some of the physical properties of glyceraldehyde-3-phosphate dehydrogenase. J. Biol. Chem., 218, 53-57.
21 Harris, J.I. and Perham, R.N. (1963) Studies on glyceraldehyde 3-phosphate dehydrogenases. Biochem. J., 89, 60-64.
22 Huitorel, P. and Pantoloni, D. (1985) Bundling of microtubules by glyceraldehyde-3-phosphate dehydrogenase and its modulation by ATP. Eur. J. Biochem., 150, 265-269.MEDLINE Abstract
23 Singh, R. and Green, M.R. (1993) Sequence-specific binding of transfer RNA by glyceraldehyde-3-phosphate dehydrogenase. Science, 259, 365-368.MEDLINE Abstract
24 Schulze, H., Schuler, A., Stuber, D., Dabeli, H., Langen, H. and Huber, G. (1993) Rat brain glyceraldehyde 3-phosphate dehydrogenase interacts with the recombinant cytoplasmatic domain of Alzheimer's -amyloid precursor protein. J. Neurochem., 60, 1915-1922.MEDLINE Abstract
25 Seipp, S. and Buselmaier, W. (1994) Isolation of glyceraldehyde 3-phosphate dehydrogenase (Gapdh) cDNA from the distal half of mouse chromosome 16: further indication of a link between Alzheimer's disease and glycolysis. Neurosci. Lett., 182, 91-94.MEDLINE Abstract
26 Meyer-Siegler, K., Mauro, D.J., Seal, G., Wurzer, J., DeRiel, J.K. and Sirover, M.A. (1991) A human nuclear uracil DNA glycosylase is the 37-kDa subunit of glyceraldehyde-3-phosphate dehydrogenase. Proc. Natl Acad. Sci. USA, 88, 8460-8464.MEDLINE Abstract
27 Ronai, Z. (1993) Glycolytic enzymes as DNA binding proteins. Int. J. Biochem., 25, 1073-1076.MEDLINE Abstract
28 Kawamoto, R.M. and Caswell, A.H. (1986) Autophosphorylation of glyceraldehyde phosphate dehydrogenase and phosphorylation of protein from skeletal muscle microsomes. Biochemistry, 25, 657-661.MEDLINE Abstract
29 Hodor, P. and Heltianu, C. (1989) Immunocytochemical localization of glyceraldehyde-3-phosphate dehydrogenase in cultured endothelial and smooth muscle cells. Microcirc. Endothelium, & Lymphatics, 5, 485-503.
30 Morgenegg, G., Winkler, G.C., Hubscher, U., Heizmann, C.W., Mous, J. and Kuenzle, C.C. (1986) Glyceraldehyde-3-phosphate dehydrogenase is a nonhistone protein and a possible activator of transcription in neurons. J. Neurochem., 47, 54-62.MEDLINE Abstract
31 Rossman, M., Liljas, A., Branden, C. and Banaszak, L. (1975) In Boyer, P.D. (ed.), The Enzymes. Academic Press, Orlando, Vol. 11, pp. 61-102.
32 Constantinides, S.M. and Deal, W.C.J. (1969) Reversible dissociation of tetrameric rabbit muscle glyceraldehyde 3-phosphate dehydrogenase into dimers or monomers by adenosine triphosphate. J. Biol. Chem., 244, 5695-56702.MEDLINE Abstract
33 Wullner, U., Young,, A.B., Penney, J.B. and Beal, M.F. (1994) 3-Nitropropionic acid toxicity in the striatum. J. Neurochem., 63, 1772-1781.MEDLINE Abstract
34 Brouillet, E., Hantraye, P., Ferrante, R.J., Dolan, R., Leroy-Willig, A., Kowall, N.W. and Beal, M.F. (1995) Chronic mitochondrial energy impairment produces selective striatal degeneration and abnormal choreiform movements in primates. Proc. Natl Acad. Sci. USA, 92, 7105-7109.MEDLINE Abstract
35 Uitti, R.J., Rajput, A.H., Ashenhurst, E.M. and Rozdilsky, B. (1985) Cyanide-induced parkinsonism: a clinicopathologic report. Neurology, 35, 921-925.MEDLINE Abstract
36 Xiao-J., L., Shi-h, L., Sharp, A.H., Nucifora, F.C., Schilling, G., Lanahan, A., Worley, P., Snyder, S.H. and Ross, C.A. (1995) A huntingtin-associated protein enriched in brain with implications for pathology. Nature, 378, 398-402.
37 Feilotter, H.E., Hannon, G.J., Ruddell, C.J. and Beach, D. (1994) Construction of an improved host strain for two hybrid screening. Nucleic Acids Res., 22, 1502-1503.MEDLINE Abstract
38 Schiestl, R.H. and Gietz, R.D. (1989) High efficiency transformation of intact yeast cells using single stranded nucleic acids as a carrier. Curr. Genet., 16, 339-346.MEDLINE Abstract
39 Breeden, L. and Nasmyth, K. (1985) Regulation of the yeast HO gene. Cold Spring Harbor Symp. Quant. Biol., 50, 643-650. MEDLINE Abstract
40 Bingham, P.M., Scott, M.O., Wang, S., McPhaul, M.J., Wilson, E.M., Garbern, J.Y., Merry, D.E. and Fischbeck, K.H. (1995) Stability of an expanded trinucleotide repeat in the androgen receptor gene in trangenic mice. Nature Genet., 9, 191-196.MEDLINE Abstract
41 Zegers, N.D., Claasen, E., Neelen, C., Mulder, E., can Laar, J.H., Voorhorst, M.M., Berrevoets, C.A., Brinkmann, A.O., van der Kwast, T.H., Ruizeveld de Winter, J.A., Trapman, J. and Boersma, W.J.A. (1991) Epitope prediction and confirmation for the human androgen receptor: generation of monoclonal antibodies for multiassay performance following the synthetic peptide strategy. Biochim. Biophys. Acta, 1073, 23.MEDLINE Abstract
42 Rose, M. and Botstein, D. (1983) Construction and use of gene fusions to lacZ([beta]-galactosidase) that are expresssed in yeast. Methods Enzymol., 101, 167-180MEDLINE Abstract
This page is maintained by OUP admin. Last updated Thu Oct 31 15:27:05 GMT 1996. Part of the OUP Journals World Wide Web service.Copyright Oxford University Press, 1996
