PROTEIN WHICH INTERACTS WITH THE HUNTINGTON'S DISEASE GENE PRODUCT, cDNA CODING THEREFOR, AND ANTIBODIES THERETO

Abstract: A protein, designated as HIP1, interacts differently with the gene product of a normal (16 CAG repeat) and an expanded (> 44 CAG repeat) HD gene. The HIP1 protein originally isolated from the yeast two-hybrid screen is encoded by a 1.2 kb cDNA, devoid of stop codons, that is expressed as a 400 amino acid polypeptide. By further screening of a human frontal cortex cDNA library, and employing the protocol for 5' Rapid Amplification of cDNA ends (RACE), a total of 4795 nucleotides (with an open reading frame of 914 amino acids) of the 10 kb message HIP1 have been isolated to date. Expression of the HIP1 protein was found to be limited to the brain, where the interaction of the HIP1 with the HD protein appears to be necessary for the association of the HD protein with the membrane or specific cytoskeletal components to render it functional. Because HIP1 interacts with expanded HD protein less well than with normal length HD, introduction of additional HIP1 or overexpression of HIP-1 can lead to increased functionality of the defective or normal HD protein. Alternatively, modified forms of the HIP1 which bind more effectively to expanded HD could be introduced to convert the expanded HD protein into a functional molecule.

Description: PROTEIN WHICH INTERACTS WITH THE HUNTINGTON'S DISEASE GENE PRODUCT, cDNA CODING THEREFOR, AND ANTIBODIES THERETO BACKGROUND OF THE INVENTION This application relates to a protein designated as HIPl which interacts with the Huntington's Disease gene product, cDNA coding for HIPl , and methods and compositions relating thereto "Interacting proteins" are proteins which associate in vivo to form specific stable complexes Non-covalent bonds, including hydrogen bonds, hydrophobic interactions and other molecular associations form between the proteins when two protein surfaces are matched or have affinity for each other This affinity or match is required for the recognition of the two proteins, and the formation of a stable interaction Protein-protein interactions are involved in the assembly of enzyme subunits, in antigen-antibody reactions, in forming the supramolecular structures of ribosomes, filaments, and viruses; in transport, and in the interaction of receptors on a cell with growth factors and hormones Huntington's disease is an adult onset disorder characterized by selective neuronal loss in discrete regions of the brain and spinal chord that lead to progressive movement disorder, personality change and intellectual decline From onset, which generally occurs around age 40, the disease progresses with worsening symptoms, ending in death approximately 18 years after onset The biochemical cause of Huntington's disease has thus far not been determined Various theories have been advanced, but each has failed to stand up to experimental evidence designed to test its validity For example, it was suggested that the selective neuronal loss could be attributed to restricted expression of mRNA or proteins in cells undergoing degeneration No obviously altered levels of mRNA transcript or protein expression has ever been observed in HD-affected tissues, however While the biochemical cause of Huntington's disease has remained elusive, a mutation in a gene within chromosome 4pl6 3 subband has been identified and linked to the disease This gene, referred to as the Huntington's Disease or HD gene, contains three repeat regions, a CAG repeat region and two CCG repeat regions Testing of Huntington's disease patients has shown that the CAG region is highly polymorphic, and that the number of CAG repeat units in the CAG repeat region is a very reliable diagnostic indicator of having inherited the gene for Huntington's disease. Thus, in control individuals and in individuals suffering from neuropsychiatric disorders other than Huntington's disease, the number of CAG repeats is between 9 and 35, while in individuals suffering from Huntington's disease the number of CAG repeats is expanded and is 36 or greater The protein product encoded by the HD gene has been localized to the cytoplasm, including to the membranes of vesicles on the brain of both normal and HD- affected individuals. To date, no differences have been observed at either the total RNA, mRNA or protein levels between normal and HD-affected individuals. Thus, the function of the HD protein and its role in the pathogenesis of Huntington's Disease remain to be elucidated. SUMMARY OF THE INVENTION We have now identified a protein, designated as HIPl , that interacts differently with the gene product of a normal (16 CAG repeat) and an expanded ( >44 CAG repeat) HD gene. The HIPl protein originally isolated from the yeast two-hybrid screen is encoded by a 1.2 kb cDNA, devoid of stop codons, that is expressed as a 400 amino acid polypeptide. By further screening of a human frontal cortex cDNA library, and employing the protocol for 5' Rapid Amplification of cDNΛ ends (RACE), a total of 4795 nucleotides (with an open reading frame of 914 amino acids) of the 10 kb message HIPl have been isolated to date. Expression of the HIPl protein was found to be limited to the brain, where the interaction of the HIPl with the HD protein appears to be necessary for the association of the HD protein with the membrane or specific cytoskeletal components to render it functional. Because HIPl interacts with expanded HD protein less well than with normal length HD, introduction of additional HIPl or overexpression of HIP l can lead to increased functionality of the defective or normal HD protein. Alternatively, modified forms of the HIPl which bind more effectively to expanded HD could be introduced to convert the expanded HD protein into a functional molecule. BRIEF DESCRIPTION OF THE DRAWING Fig. 1 graphically depicts the amount of interaction between HIPl and Huntingtin proteins with varying lengths of polyglutamine repeat. DETAILED DESCRIPTION OF THE INVENTION The HI l protein which interacts with the HD gene product was identified using the yeast two-hybrid system described in US Patent No. 5,283, 173 which is incorporated herein by reference. Briefly, thi system utilizes two chimeric genes or plasmids expressible in a yeast host. The yeast host is selected to contain a detectable marker gene having a binding site for the DNA binding domain of a transcriptional activator. The first chimeric gene or plasmid encodes a DNA-binding domain which recognizes the binding site of the selectable marker gene and a test protein or protein fragment. The second chimeric gene or plasmid encodes for a second test protein and a transcriptional activation domain. The two chimeric genes or plasmids are introduced into the host cell and expressed, and the cells are cultivated. Expression of the detectable marker gene only occurs when the gene product of the first chimeric gene or plasmid binds to the DNA binding domain of the detectable marker gene, and a transcriptional activation domain is brought into sufficient proximity to the DNA- binding domain, an occurrence which is facilitated by protein-protein interactions between the first and second test proteins. By selecting for cells expressing the detectable marker gene, those cells which contain chimeric genes or plasmids for interacting proteins can be identified, and the gene can be recovered and identified. In testing for Huntington Interacting Proteins, several different plasmids were prepared containing portions of the HD gene. The first four, identified as 16pGBT9, 44pGBT9, 80pGBT9 and 128pGBT9, were GAL4 DNA binding domain-HD in-frame fusions containing nucleotides 314 to 1955 (amino acids 1-540) of the published HD cDNA sequences cloned into the vector pGBT9 (Clontech). These plasmids contain a CAG repeat region of 16, 44, 80 and 128 glutamine-encoding repeats, respectively. A clone (DMK BamHIpGBT9) was made by fusing acDNA encoding the first 544 amino acids of the myotonic dystrophy gene (a gift from R. Komeluk) in-frame with the GAL4-DNA BD of pGBT9 and was used as a negative control. These plasmids have been used to identify and characterize HIP 1 , two additional HD-interacting proteins, HIP2 and HIP3 proteins, and can be further used for the identification of additional interacting proteins, and for tests to refine the region on the protein in which the interaction occurs. Thus, a first aspect of the invention is these four plasmids, and the use of this plasmids in identifying HD-interacting proteins. Furthermore, it will be appreciated that the GAL4 DNA-binding and activating domains are not the only domains which can be used in the yeast two-hybrid assay. Thus, in a broader sense, the invention encompasses any chimeric genes or plasmids containing nucleotides 314 to 1955 of the HD gene together with an activating or DNA-binding domain suitable for use in the yeast one, two- or three-hybrid assay for proteins critical in either binding to the HD protein or responsible for regulated expression of the HD gene. After introducing the plasmids into Y190 yeast host cells, transforming the host cells with an adult human brain Matchmaker 1" " (Clontech) cDNA library coupled with a GAL4 activating domain, and selecting for the expression of two detectable marker genes to identify clones containing genes for interacting proteins, the activating domain plasmids were recovered and analyzed. As a result of this analysis, three different cDNA fragments were identified as encoding for HD-interacting proteins and designated as HIPl, HIP2 and HIP3 The sequences of HIP l and HIP3 are given in Seq. ID Nos 1 and 3 The polypeptides which each encodes are given by Seq. ID Nos. 2 and 4. Further investigation of the HIPl cDNA resulted in the characterization of an additional region of cDNA totaling 4795 bases and a corresponding protein, the sequences of which are given by Seq ID Nos. 5 and 6, respectively The cDNA molecules, particularly those encoding portions of HIPl , can be explored using oligonucleotide probes for example for amplification and sequencing, ln addition, oligonucleotide probes complementary to the cDNA can be used as diagnostic probes to localize and quantify the presence of HIPl DNA. Probes of this type with a one or two base mismatch can also be used in site-directed mutagenesis to introduce variations into the HIPl sequence which may increase. Thus, a further aspect of the present invention is an oligonucleotide probe, preferably having a length of from 15-40 bases which specifically and selectively hybridizes with the cDNA given by Seq. ID No. 1 or 5 or a sequence complemen- tary thereto As used herein, the phrase "specifically and selectively hybridizes with" the cDNA refers to primers which will hybridize with the cDNA under stringent hybridization conditions DNA sequencing of the HIPl cDNA initially isolated from the yeast two-hybrid screen revealed a 1 2 kb cDN A that shows no significant degree of nucleic acid identity with any stretch of DNA using the blastn program at ncbi (blast@ncbi.nl m nih.gov) When the entire HIP l cDNA sequence (SEQ ID NO 5) is translated into a polypeptide, the entire HIPl cDNA coding (nucleotides 328-3069) is observed to be devoid of stop codons, and to produce a 914 amino acid polypeptide A polypeptide identity search revealed an identity match over the entire length of the protein (46% conservation) with that of a hypothetical protein from C. elegans (ZK370.3 protein, C. elegans cosmid ZK370) This C. elegans protein shares iden¬ tity with the mouse talin gene, which encodes a 217 kDa protein implicated with maintain¬ ing integrity of the cytoskeleton. It also shares identity with the SLA2/MOP2/ END4 gene from Saccharomyces cerevisiae, which is known to code for an essential cytoskeletal associated gene required for the accumulation and or maintenance of plasma membrane H + - ATPase on the cell surface. When pairwise comparisons are performed between HIPl and the C. elegans ZK370.3 protein (Genpept accession number celzk370.3), it shows 26% complete identity and an overall 46% level of conservation. Comparative analysis between HIPl and SLA2/MOP2/ END4 (EMBL accession number Z2281 1) demonstrate similar conservation (20% identity, 40% conservation). HIP2 is a 2.0 kb cDNA that encodes all but the 5 '-most 33 amino acids of human E2 25k ubiquitin conjugating enzyme. The resulting peptide has 100% identity with the previously characterized bovine E2 25k protein. The cDNA has 95 % nucleotide identity with the bovine cDNA. Ubiquitin-conjugating enzyme is an important component in ubiquitin-mediated protein degradation pathways. No difference in the strength of the interaction between HIP2 and HD constructs containing either 44 or 15 CAG repeats is detected using a quantitative β- galactosidase assay. The expression pattern of HIP2 (E2 25k ) in the various pans of the brain and nervous system appears to follow the specific neuropathology observed in HD, although there does not appear to be any difference in expression levels between HD- affected and HD-non-affected individuals. The third cDNA encoding an HD-interacting protein is a 537 bp cDNA coding for 187 ammo acids. A search of known DNA databases did not identify the sequence homology with any known genes. However, when a protein search was per¬ formed using the blatsp server, a strong identity between HIP3 and ankyπn-related proteins was observed. The strongest identity was with the D2021.8 gene product of C. elegans, an uncharacterized gene, but there is also a 41 % identity with AKR1 , a yeast ankyπn repeat- containing protein Furthermore, when analogous structures with charge conservation over the same amino acid stretch are considered, there is 70% protein identity. HIP3 also shares approximately 60% amino acid conservation with human brain specific ankyπns (ankyπn B and ankyπn C). Thus, it is reasonable to conclude that HIP3, like known ankyπns, is a cytoskeletal protein, and may be involved, like previously characterized ankyπns in promoting interactions between the membrane skeleton and other membrane proteins. Further exploration of these three HD interacting proteins revealed several important facts about HIPl that implicate it in a significantly in the pathogenesis of Huntington's Disease. First, as shown in Fig. 1 , it was found that the strength of the interaction between HD protein and HIPl is dependent on the number of CAG repeats. Second, it was found that expression of the HIPl protein is not ubiquitous, but is limited to brain tissue. The highest amounts of expression are in the cortex, with lower levels being seen in the cerebellum, caudate and putamen Both HIPl and HIP3 appear to be proteins which are involved in the maintaining the structural integrity of the cytoskeleton and various components of the cellular membrane, including microtubules and focal adhesions. Based upon this, the HD protein may be associated as part of the cytoskeletal matrix in cells where it is expressed, and our work supports the conclusion that binding of HIPl to the HD protein is necessary for the functional incoφoration of the HD protein into the cell membrane. In this circumstance, the larger polyglutamine tract in huntingtin has a decreased ability for an H1P1-HD interaction. This decreased affinity for each other disrupts the normally strong HD-HIP1 -cytoskeletal anchoring association. Further, the HIP1-HD interaction may be a critical interaction at the membranes of synaptic vesicles and a decrease in the affinity of HIPl for huntingtin may affect protein trafficking or membrane organization throughout the neuron. Finally, we have demonstrated that HIPl and HD are both found in the Triton X-100 insoluble membrane compartment of the cell, therefore, a decreased interaction between HIPl and huntingtin may allow an abnormally subtle amount of huntingtin to be found in subcelluiar compartments in which it is normally found. As a result of all three of these phenomenon, increased apoptosis can occur in specific neurons within the striatum. This increase in apoptosis arises from an increased susceptibility of polyglutamine-expanded huntingtin to cleavage by apopain, and because more of the expanded forms of the HD protein may be available for cleavage (and subsequent apoptosis) due to the fact they are not as tightly associated at the HD-HIP1- cytoskeletal complex. This understanding of a biochemical basis for the pathogenesis of Huntington's Disease opens the doorway to a therapeutic method to ameliorate the pathology in patients expressing huntingtin protein with expanded polyglutamine tracts. In accordance with the method, the patient is treated to increase the amount of HIPl or an equivalent polypeptide which interacts less well with expanded Huntingtin than with Huntingtin having a CAG repeat region containing 15 to 35 repeats and facilitates the incorporation of Huntingtin into brain cell membranes. Increasing expression of HIPl or an equivalent polypeptide can be accomplished using gene therapy approaches. In general, this will involve introduction of DNA encoding HIPl in an expressable vector into the brain cells. Vectors which have been shown to be suitable expression systems in mammalian cells include the heφes simplex viral based vectors: pHSVl (Geller et al. Proc. Natl. Acad. Sci 87:8950-8954 (1990)); recombinant retroviral vectors: MFG (Jaffee et al. Cancer Res. 53:2221-2226 (1993)); Moloney-based retroviral vectors: LN, LNSX, LNCX, LXSN (Miller and Rosman Bio'echniques 7:980-989 (1989)); vaccinia viral vector: MVA (Sutter and Moss Proc. Natl. Acad. Sci. 89: 10847-10851 (1992)); recombinant adenovirus vectors : pJM 17 (Ali et al Gene Therapy 1 :367-384 (1994)), (Berkner K. L. Biotechniques 6:616-624 1988): second generation adenovirus vector: DE1/DE4 adenoviral vectors (Wang and Finer Nature Medicine 2:714-716 (1996) ); and Adeno-associated viral vectors: AAV/Neo (Muro-Cacho et al. J. Immunotherapy 11 :231-237 (1992)). Delivery of retroviral vectors to brain and nervous system tissue has been described in US Patents Nos. 4,866,042, 5,082,670 and 5,529,774, which are incorporated herein by references. These patents disclose the use of cerebral grafts or implants as one mechanism for introducing vectors bearing therapeutic gene sequences into the brain, as well as an approach in which the vectors are transmitted across the blood brain barrier. In addition to increasing the amount of HIPl present in brain cells of affected individuals, HD lethal phenotype may be rescued by coexpression of a HIPl and normal sized HD protein within the same cell, specifically within neurons. The over¬ expression of the normal HD protein and the presence of excess HIPl in the cell may be able to override the damaging effects of a decreased interaction between HIPl and an expanded form of the HD protein. Therefore, a "normal state" of interaction of HD with HIPl will rescue the cell from premature apoptotic death. Thus, a therapeutically desirable mammalian expression vector may include both a region encoding HIPl and a region encoding normal (less than 35 repeats) HD protein. To further illustrate the methods of making the materials which are the subject of this invention, and the testing which has established their utility, the following non-limiting experimental procedures are provided. EXAMPLE 1 IDENTIFICATION OF INTERACTING PROTEINS GAL4-HD cDNA constructs An HD cDNA construct (44pGBT9), with 44 CAG repeats was generated encompassing amino acids 1 - 540 of the published HD cDNA . This cDNA fragment was fused in frame to the GAL4 DNA-binding domain (BD) of the yeast two-hybrid vector pGBT9 (Clontech). Other HD cDNA constructs, 16pGBT9, 80pGBT9 and 128pGBT9 were constructed, identical to 44pGBT9 but included only 16, 80 or 128 CAG repeats, respectively. Another clone (DMKDBamHIpGBT9) containing the first 544 amino acids of the myotoπic dystrophy gene (a gift from R. Korneluk) was fused in-frame with the GAL4-DNA BD of pGBT9 and was used as a negative control. Plasmids expressing the GAL4-BDRAD7 (D. Gietz, unpublished) and SIR3 were used as a positive control for the β-galactosidase filter assay. The clones IT15-23Q, IT15-44Q and HAP1 were generous gifts from Dr. C. Ross. These clones represent a previously isolated huntingtin interacting protein that has a higher affinity for the expanded form of the HD protein. Yeast strains, transformations and β-galactosidase assays The yeast strain Y190 (MATa leu2-3,l 12, ura3-52, iτpl -901, his3-Δ200, ade2-101 , gal4Δgal80Δ, URA3 GAL-lacZ, LYS2 GAL-HIS3,cyc r ) was used for all transformations and assays Yeast transformations were performed using a modified lithium acetate transformation protocol and grown at 30 C using appropriate synthetic complete (SC) dropout media The β-galactosidase chromogenic filter assays were performed by transfer- ring the yeast colonies onto Whatman filters. The yeast cells were lysed by submerging the filters in liquid nitrogen for 15-20 seconds. Filters were allowed to dry at room tempera¬ ture for at least five minutes and placed onto filter paper presoaked in Z-buffer (100 mM sodium phosphate (pH7.0) 10 mM KCl, 1 mM MgSO 4 ) supplemented with 50 M 2-mercaptoethanol and 0.07 mg/ml 5-bromo-4-chloro-3-indolyi β-D-galactoside (X-gal). Filters were placed at 37 C for up to 8 hours. Yeast two-hybrid screening for huntingtin interacting protein (HIP) cDNAs from an human adult brain Matchmaker™ cDNA library (Clontech) was transformed into the yeast strain Yl 0 already harboring the 44pGBT9 construct The transformants were plated onto one hundred 150 mm x 15 mm circular culture dishes containing SC media deficient in Trp, Leu and His The herbicide 3-amιno-triazole (3 -AT) (25mM) was utilized to limit the number of false His+ positives (31) The yeast transformants were placed at 30 C for 5 days and β-galactosidase filter assays were performed on all colonies found after this time, as described above, to identify β-galactosidase+ clones Primary His+/β-galactosidase+ clones were then orderly patched onto a grid on SC -Trp/-Leu/-His (25 mM 3AT) plates and assayed again for His+ growth and the ability to turn blue with a filter assay. Secondary positives were identified for further analysis. Proteins encoded by positive cDNAs were designated as HIPs (Huntingtin Interactive Proteins). Approximately 4.0 x I O 7 Tφ/Leu auxotrophic transformants were screened and of 14 clones isolated 12 represented the same cDNA (HIPl), and the other 2 cDNAs, HIP2 and HIP3 were each represented only once. The HIP cDNA plasmids were isolated by growing the His + /β- galactosidase+ colony in SC -Leu media overnight, lysing the cells with acid-washed glass beads and electroporating the bacterial strain, KC8 (leuB auxotrophic) with the yeast lysate. The KC8 ampicillin resistant colonies were replica plated onto M9 (-Leu) plates. The plasmid DNA from M9+ colonies was transformed into DH5-a for further manipulation. EXAMPLE 2 CONFIRMATION OF INTERACTIONS The HIP1-GAL4-AD cDNA activated both the lac-Z and His reporter genes in the yeast strain Y190 only when co-transformed with the GAL4-BD-HD construct, but not the negative controls (Figure 1) of the vector alone or a random fusion protein of the myotonin kinase gene. In order to assess the influence of the polyglutamine tract on the interaction between HIPl and HD, semi-quantitative β-galactosidase assays were performed. GAL4-BD-HD fusion proteins with 16, 44, 80 and 128 glutamine repeats were assayed for their strength of interaction with the GAL4-AD-HIP1 fusion protein. Liquid β-galactosidase assays were performed by inoculating a single yeast colony into appropriate synthetic complete (SC) dropout media and grown to OD600 0.6-1.5. Five millilitres of overnight culture was pelleted and washed once with 1 ml of Z-Buffer, then resuspended in 100 ml Z-Buffer supplemented with 38 mM 2-mercapto- ethanol, and 0.05 % SDS. Acid washed glass beads ( " 100 ml) were added to each sample and vortexed for four minutes, by repeatedly alternating a 30 seconds vortex, with 30 seconds on ice. Each sample was pelleted and 10 ml of lysate was added to 500 ml of lysis buffer. The samples were incubated in a 30 C waterbath for 30 seconds and then 100 ml of a 4 mg/ml o-nitrophenyl b-D galactopyranoside (ONPG) solution was added to each tube. The reaction was allowed to continue for 20 minutes at 30 C and stopped by the addition of 500 ml of 1 M Na 2 CO 3 and placing the samples on ice. Subsequently, OD420 was taken in order to calculate the β-galactosidase activity with the equation 1000 x OD420/(t x V x OD600) where t is the elapsed time (minutes) and V is the amount of lysate used. The specificity of the HΪP1-HD interaction can be observed using the chromogenic filter assay. Only yeast cells harboring HIPl and HD activate both the HIS and lacZ reporter genes in the Y190 yeast host. The cells that contain the HIPl with HD constructs with 80 or 128 CAG repeats turn blue approximately 45 minutes after the cells with the smaller sized repeats (16 or 44). No difference in the β-galactosidase activity was observed between the 16 and 44 repeats or between the 80 and 128 repeats. However, a significant difference (p < 0.05) in activity is seen between the smaller repeats (16 and 44) and the larger repeats (80 and 128). (Figure 1) EXAMPLE 3 DNA SEQUENCING. cDNA ISOLATION AND 5' RACE Oligonucleotide primers were synthesized on an ABI PCR-mate oligo- synthesizer. DNA sequencing was performed using an ABI 373 fluorescent automated DNA sequencer. The HIP cDNAs were confirmed to be in-frame with the GAL4-AD by sequencing across the AD-HIP1 cloning junction using an AD oligonucleotide (5'GAA GAT ACC CCA CCA AAC3'). Subsequently, primer walking was used to determine the remaining sequences. A human frontal cortex >4.0 kb cDNA library (a gift from S. Montal) was screened to isolate the full length HIPl gene. Fifty nanograms of a 558 base pair Eco RI fragment from the original HIPl cDNA was radioactively labeled with |α 32 P]-dCTP using nick-translation and the probe allowed to hybridized to filters containing > 105 pfu/ml of the cDNA library overnight at 65 C in Church buffer (see Northern blot protocol). The filters were washed at 65 C for 10 minutes with 1 X SSPE, 15 minutes at 65 C with 1 X SSPE and 0.1 % SDS, then for thirty minutes and fifteen minutes with 1 X SSPE and 0.1 % SDS. The filters were exposed to X-ray film (Kodak. XAR5) overnight at -70 C. Primary positives were isolated and replated and subsequent secondary positives were hybridized and washed as for the primary screen. The resulting positive phage were converted into plasmid DNA by conventional methods (Stratagene) and the cDNA isolated and sequenced. In order to obtain the most 5' sequence of the HIPl gene, a Rapid Amplification of cDNA Ends (RACE) protocol was performed according to the manufacturers recommendations (BRL). First strand cDNA was synthesized using the oligo HIP1-242R (5' GCT TGA CAG TGT AGT CAT AAA GGT GGC TGC AGT CC 3'). After dCTP tailing the cDNA with terminal deoxy transferase, two rounds of 35 cycles (94 C 1 minute; 53 C 1 minute; 72 C 2 minutes) of PCR using HIP1-R2 (5' GGA CAT GTC CAG GGA GTT GAA TAC 3') and an anchor primer (5 * (CUA)4 GGC CAC GCG TCG ACT AGT ACG GGI IGG GII GGG IIG3') (BRL) were performed. The subsequent 650 base pair PCR product was cloned using the TA cloning system (Invitrogen) and sequenced using T3 and T7 primers. Sequences ID Nos. 1 and 5 show the sequence of the HIPl cDNAs obtained. EXAMPLE 4 DNA AND AMINO ACID ANALYSES Overlapping DNA sequence was assembled using the program MacVector and sent via email or Netscape to the BLAST server at NIH (http://www.ncbi.nlm.nih.gov) to search for sequence similarities with known DNA (blastn) or protein (tblastn) sequences. Amino acid alignments were performed with the program Clustalw. EXAMPLE 5 FISH DETECTION SYSTEM AND IMAGE ANALYSIS The HIPl cDNA isolated from the two-hybrid screen was mapped by fluorescent in situ hybridization (FISH) to normal human lymphocyte chromosomes counterstained with propidium iodide and DAPI. Biotinylated probe was detected with avidin-fluorescein isothiocyanate (FITC). Images of metaphase preparations were captured by a thermoelectrically cooled charge coupled camera (Photometries). Separate images of DAPI banded chromosomes and FITC targeted chromosomes were obtained. Hybridization signals were acquired and merged using image analysis software and pseudo colored blue (DAPI) and yellow (FITC) as described and overlaid electronically. This study showed that HIPl maps to a single genomic locus at 7ql l .2. EXAMPLE 6 NORTHERN BLOT ANALYSIS RNA was isolated using the single step method of homogenization in guanidinium isothiocyante and fractionated on a 1.0% agarose gel containing 0.6 M formaldehyde. The RNA was transferred to a hybond N -membrane (Amersham) and crosslinked with ultraviolet radiation. Hybridization of the Northern blot with b-actin as an internal control probe provided confirmation that the RNA was intact and had transferred. The 1.2 kb HIPl cDNA was labeled using nick translation and incorporation of cr^P-dCTP. Hybridization of the original 1.2 kb HIPl cDNA was carried out in Church buffer (0.5 M sodium phosphate buffer, pH 7.2, 2.7 % sodium dodecyl sulphate, 1 M EDTA) at 55 C over¬ night. Following hybridization, Northern blots were washed once for 10 minutes in 2.0 X SSPE, 0.1 % SDS at room temperature and twice for 10 minutes in 0.15 X SSPE, 0. 1 % SDS. Autoradiography was carried our from one to three days using Hyperfilm (Amersham) film at -70 C. Analysis of the levels of RNA levels of HIPl by Northern blot data revealed that the 10 kilo base HIPl message is present in all tissue assessed. However, the levels of RNA are not uniform, with brain having highest levels of expression and peripheral tissues having less message. No apparent differences in RNA expression was noted between control samples and HD affected individuals. EXAMPLE 7 TISSUE LOCALIZATION OF HIPl Tissue localization of HIPl was studied using a variety of techniques as described below. Subcellular distribution of HIP-1 protein in adult human and mouse brain Biochemical fractionation studies revealed the HIPl protein was found to be a membrane-associated protein. No immunoreactivity was seen by Western blotting in cytosolic fractions, using the anti-HIPl-pepl polyclonal antibody. HIPl immunoreactivity was observed in all membrane fractions including nuclei (PI), mitochondria and synapto- somes (P2), microsomes and plasma membranes (P3). The P3 fraction contained the most HIPl compared to other membrane fractions. HIPl could be removed from membranes by high salt (0.5M NaCl) buffers indicating it is not an integral membrane protein, however, since low salt (0.1- 0.25M NaCl) was only able to partially remove HIPl from membranes, its membrane association is relatively strong. The extraction of P3 membranes with the non-ionic detergent, Triton X-100 revealed HIPl to be a Triton X-100 insoluble protein. This characteristic is shared by many cytoskeletal and cytoskeletal-associated membrane proteins including actin, which was used as a control in this study. The biochemical characteristics of HIPl described were found to be identical in mouse and human brain and was the same for both forms of the protein (both bands of the HIPl doublet). HIPl co-localized with huntingtin in the P2 and P3 membrane fractions, including the high-salt membrane extractions, as well as in the Triton X-100 insoluble residue. The subcellular distribution of HIPl was unaffected by the expression of polyglutamine-expanded huntingtin in transgenic mice and HD patient brain samples. The localization of HIPl protein was further investigated by immunohisto¬ chemistry in normal adult mouse brain tissue. Immunoreactivity was seen in a patchy, reticular pattern in the cytoplasm, appeared excluded from the nucleus and stained most intensely in a discontinuous pattern at the membrane. These results are consistent with the association of HIPl with the cytoskeletal matrix and further indicate an enrichment of HIPl at plasma membranes. Immunoreactivity occurred in all regions of the brain, including cortex, striatum, cerebellum and brainstem, but appeared most strongly in neurons and especially in cortical neurons. As described previously, huntingtin immunoreactivity was seen exclusively and uniformly in the cytosol. The in situ hybridization studies showed HIPl mRNA to be ubiquitously and generally expressed throughout the brain. This data is consistent with the immunohisto- chemical results and was identical to the distribution pattern of huntingtin mRNA in transgenic mouse brains expressing full-length human huntingtin. Protein Preparation And Western Blotting For Expression Studies Frozen human tissues were homogenized using a Polytron in a buffer containing 0.25M sucrose, 20mM Tris-HCl (pH 7.5), lOmM EGTA, 2mM EDTA supplemented with lOug/ml of leupeptin, soybean trypsin inhibitor and lmM PMSF, then centrifuged at 4,000φm for 10' at 4 C to remove cellular debris. 100-150ug/lane of protein was separated on 8% SDS-PAGE mini-gels and then transferred to PVDF membranes. Huntingtin and HIPl were electroblotted overnight in Towbin's transfer buffer (25 mM Tris-HCl, 0.192M glycine, pH8.3, 10% methanol) at 30V onto PVDF membranes (Immobilon-P, Millipore) as descπbed (Towbin et al, Proc. Nat 'l Acad. Sci. (USA) 76: 4350-4354 (1979)) Membranes were blocked for 1 hour at room temperature in 5 % skim milk/ TBS (lOmM Tris-HCl, 0.15M NaCl, pH7.5). Antibodies against huntingtin (pAb BKP1 , 1 :500), actin (mAb A-4700, Sigma, 1 :500) or HIPl (pAb HIP-pepI , 1 :200) were added to blocking solution for 1 hour at room temperature. After 3 x 10 minutes washes in TBS-T (0.05 % Tween-20/TBS), secondary Ab (horseradish peroxidase conjugated IgG, Biorad) was applied in blocking solution for 1 hour at room temperature. Membranes were washed and then incubated in chemiluminescent ECL solution and visualized using Hyperfilm-ECL film (Amersham) Generation of Antibodies The generation of huntingtin specific antibodies GHM 1 and BKP1 is des¬ cπbed elsewhere (Kalchman, et al. , J. Biol. Chem. 271 : 19385-19394 (1996)). The HIPl peptide (VLEKDDLMDMDASQQN, a.a. 76-91 of Seq. ID No. 2) was synthesized with Cys on the N-terminus for the coupling, and coupled to Keyhole limpet hemocyanin (KLH) (Pierce) with succinimidyl 4-(N-maleιmιdomethyl) cyclohexame-1 -carboxylate (Pierce). Female New Zealand White rabbits were injected with HIPl peptide-KLH and Freund's adjuvant Antibodies against the HIPl peptide were puπfied from rabbit sera using affinity column with low pH elution. Affinity column was made by incubation of HIPl peptide with activated thio-Sepharose (Pharmacia). Western blotting of various peripheral and brain tissues were consistent with the RNA data. The HIPl protein levels observed was not ubiquitous. The protein expression is limited to brain tissue, with highest amounts seen in the cortex and lower levels seen in the cerebellum and caudate and putamen. More reg io- specific analysis of HIPl expression in the brain revealed no differential expression pattern in affected individuals when compared to normal controls, with highest levels of expression seen in both controls and HD patients in the cortical regions. EXAMPLE 8 CO-IMMUNOPRECIPITATION OF HIPl WITH HUNTINGTIN Confirmation of the HD-HIP1 interaction was performed using coimmuno- prepitation as follows. Control human brain (frontal cortex) lysate was prepared in the same manner as for subcellular localization study. Prior to immunoprecipitation, tissue lysate was centrifuged at 5000 φm for 2 minutes at 4 C, then the supernatant was pre-cleared by the incubated with excess amount of Protein A-Sepharose for 30 minutes at 4 C, and centrifuged at the same condition. Fifty microlitres of supernatant (500 mg protein) was incubated with or without antibodies (10 ug of anti-huntingtin GHM l (Kalchman, et al. 1996) or anti-synaptobrevin antibody) in the total 500 ul of incubation buffer (20mM Tris-Cl (pH7.5), 40mM NaCl, l mM MgCI 2 ) for 1 hour at 4 C. Twenty microlitres of Protein A-Sepharose (1 : 1 suspension, for GHM l and no antibody control) or Protein G-Sepharose (for anti-synaptobrevin antibody; Pharmacia) was added and incubated for 1 hour at 4 C. The beads were washed with washing buffer (incubation buffer containing 0.5 % Triton X-100) three times. The samples on the beads were separated using SDS-PAGE (7.5 % acrylamide) and transferred to PVDF membrane (Immobilon-P, Millipore). The membrane was cut at about 150 kDa after transfer for Western blotting (as described above). The upper piece was probed with anti-huntingtin BKP1 (1/1000) and lower piece with anti-HIPl antibody (1/300). The results showed that when an anti-HIPl polyclonal antibody was immunoreacted against a blot containing the GHM l immunoprecipitates from the brain lysate a doublet was observed at approximately 100 kDa was. When GHM l was immuno- reacted against the same immunoprecipitate the 350 kDa HD protein was also seen The specificity of the HD-HIP1 interaction is seen as no immunoreactive bands seen are as a result of the proteins adsorbing to the Protein-A-Sepharose (Lysate + No Antibody) or when a random, non related antibody (Lysate + anti-Synaptobrevin) is usυd as the immunoprecipitating antibody. EXAMPLE 9 Subcellular fractionation of brain tissue Cortical tissue (20-100 mg/ml) was homogenized, on ice, in a 2 ml pyrex-teflon IKA-RW15 homogenizer (Tekmar Company) in a buffer containing 0.303M sucrose, 20mM Tris-HCl pH 6.9, l mM MgCl 2 , 0.5mM EDTA, lmM PMSF, lmM leupeptin, soybean trypsin inhibitor and l mM benzamidine (Wood et al., Human Molec. Genet. 5. 481 -487 (1996)). Crude membrane vesicles were isolated by two cycles of a three-step differ¬ ential centrifugation protocol in a Beckman TLA 120.2 rotor at 4 C based on the methods of Wood et al (1996). The first step precipitated cellular debris and nuclei from tissue ho ogenates for 5 minutes at 1300 x g (Pl). The 1300 x g supernatant was subsequently centrifuged for 20 minutes at 14 000 x g to isolate synaptosomes and mitochondria (P2). Finally, microsomal and plasma membrane vesicles were collected by a 35 minute centrifugation at 142 000 x g (P3). The remaining supernatant was defined as the cytosolic fraction. High salt extraction of membranes Aliquots of P3 membranes were twice suspended at 2mg/ ml in 0.5M NaCl, lOmM Tris-HCl, 2mM MgCL, pH7.2, containing protease inhibitors (see above). The same buffer without NaCl was used as a control. The membrane suspensions were incubated on ice for 30 minutes and then centrifuged at 142 000 x g for 30 minutes. Extraction of cytoskeletal and cytoskeletal-associated proteins. To extract cytoskeletal proteins, crude membrane vesicles from the P3 fraction membrane were suspended in a volume of Triton X-100 extraction buffer to give a protein: detergent ratio of 5: 1. The composition of the Triton X-100 extraction buffer was based on the methods of Arai et al., J. Neuroscience 38: 348-357 (1994) and contained 2 % Triton X-100, lOmM Tris-HCl, 2mM MgCl 2 , lmM leupeptin, soybean trypsin inhibitor, PMSF and benzamidine. Membrane pellets were suspended by hand with a round-bottom teflon pestle, and placed on ice for 40 minutes. Insoluble cytoskeletal matrices were precipitated for 35 minutes at 142 000 x g in a Beckman TLA 120.2 rotor. The supernatant was defined as non-cytoskeletal-associated membrane or membrane- -associated protein and was removed. The remaining pellet was extracted with Triton X-100 a second time using the same conditions. We defined the final pellet as cytoskeletal and cytoskeletal-associated protein. Solubilization of protein and analysis by SDS-PAGE and Western Blotting Membrane and cytoskeletal protein was solubilized in a minimum volume of 1 % SDS, 3M urea, 0. lmM dithiothreitol in TBS buffer and sonicated. Protein concen- tration was determined using the BioRad DC Protein assay and samples were diluted at least 1 X with 5 X sample buffer (250mM Tris-HCl pH 6.8, 10% SDS, 25 % glycerol, 0.02 % bromophenol blue and 7% 2-mercaptoethanol) and were loaded on 7.5 % SDS-PAGE gels (Bio-Rad Mini-PROTEIN II Cell system) without boiling. Western blotting was performed as described above. Immunohistochemistry Brain tissue was obtained from a normal C57BL/6 adult (6 months old) male mouse sacrificed with chloroform then perfusion-fixed with 4% v/v paraformaldehyde/0.01 M phosphate buffer (4% PFA). The brain tissues were removed, immersion fixed in 4% PFA for 1 day, washed in 0.01 M phosphate buffered saline, pH 7.2 (PBS) for 2 days, and then equilibrated in 25 % w/v sucrose PBS for 1 week. The samples were then snap-frozen in Tissue Tek molds by isopentane cooled in liquid nitrogen. After warming to -20 C, frozen blocks derived from frontal cortex, caudate/putamen, cerebellum and brainstem were cut into 14 mm sections for immunohistochemistry. Following washing in PBS, the tissue sections were blocked using 2.5 % v/v normal goat serum for 1 hour at room temperature. Primary antibodies diluted with PBS were applied to sections overnight at 4 C. Optimal dilutions for the polyclonal antibodies BKP1 and HIPl were 1 :50 Using washes of 3 x 5 minutes in PBS at room temperature, sections were sequentially incubated with biotinylated secondary antibody and then an avidin-biotin complex reagent (Vecta Stain ABC Kit, Vector) for 60 minutes each at room temperature. Color was developed using 3-3'-dιamιnobenzιdιne tetrahydrocholoπde and ammonium nickel sulfate For controls, sections were treated as described above except that HIPl antibody aliquots were preabsorbed with an excess of HIPl peptide as well as a peptide unrelated to HIPl prior to incubation with the tissue sections. In situ hybridization In situ hybridization was performed as previously described with some modification .Suzuki et al, BBRC 2\9. 708-713 (1996)). The RNA probes were prepared using the plasmid gtl49 (Lin, B., et al. , Human Molec. Genet. 2. 1541-1545 (1994)) or a 558 subclone of HIPl . The anti-sense and sense single-stranded RNA probes were synthesized using T3 and T7 RNA polymerases and the In Vitro Transcription Kit (Clontech) with the addition of [ 35 SJ-CTP (Amersham) to the reaction mixture. Sense RNA probes were used as negative controls. For HIPl studies normal C57BL/6 mice were used Huntingtin probes were tested on two different transgenic mouse strains expressing full-length huntingtin, cDNA HD10366(44CAG) C57BL/6 mice and YAC HD10366(18CAG) FVB/N mice. Frozen brain sections (lOum thick) were placed onto silane coated slides under RNase-free conditions. The hybridization solution contained 40% w/v formamide, 0.02M Tris-HCl (pH 8.0), 0.005M EDTA, 0.3 M NaCl, 0.01M sodium phosphate (pH 7.0), lx Denhardt's solution, 10% w/v dextran sulfate (pH 7.0), 0.2 % w/v sarcosyl, yeast tRNA (500mg/ml) and salmon sperm DNA (200mg/ml). The radiolabelled RNA probe was added to the hybridization solution to give 1 x 106 cpm/200 ul/ section. Sections were covered with hybridization solution and incubated on formamide paper at 65 C for 18 hours. After hybridization, the slides were washed for 30 minutes sequentially with 2x SSC, lx SSC and high stringency wash solution (50% formamide, 2x SSC and 0.1 M dithiothreitol) at 65 C, followed by treatment with Rnase A (1 mg/ml) at 37 C for 30 minutes, then washed again and air-dried. The slides were first exposed on autoradiographic film (b-max, Amersham, UK) for 48 hours and developed for 4 minutes in Kodak D-19 followed by a 5 minute fixation in Fuji-fix. For longer exposures, the slides were dipped in autoradiographic emulsion (50% w/v in distilled water, NR-2, Konica, Japan), air-dried and exposed for 20 days at 4 C then developed as described. Sections were counterstained with methyl green or Giemsa solutions. SEQUENCE LISTING ( 1 ) GENERAL INFORMATION (i) APPLICANT Kalchman, Michael Goldberg, Paul Hayden Michael R (ii) TITLE OF INVENTION Protein Which Interacts with the Huntington's Disease Gene Product, cDNA Coding Therefor, and Antibodies Thereto (iii) NUMBER OF SEQUENCES 8 (iv) CORRESPONDENCE ADDRESS (A) ADDRESSEE Oppedahl & Larson (B) STREET 1992 Commerce Street Suite 309 (C) CITY Yorktown (D) STATE NY (E) COUNTRY USA (F) ZIP 10598 (v) COMPUTER READABLE FORM (A) MEDIUM TYPE Diskette, 3 50 inch, 1 44 Kb storage (B) COMPUTER IBM Compatible (C) OPERATING SYSTEM MS DOS 5 0 (D) SOFTWARE WordPerfect (vi) CURRENT APPLICATION DATA (A) APPLICATION NUMBER (B) FILING DATE (C) CLASSIFICATION (viii) ATTORNEY/AGENT INFORMATION (A) NAME Larson, Marina T (B) REGISTRATION NUMBER 32038 (C) REFERENCE DOCKET NUMBER UBC P-013 (ix) TELECOMMUNICATION INPORMATION (A) TELEPHONE (914) 245-3252 (B) TELEFAX (914) 962-4330 (2) INFORMATION FOR SEQ ID NO 1 (i) SEQUENCE CHARACTERISTICS (A) LENGTH 1 164 (B) TYPE nucleic acid (C) STRANDEDNESS single (D) TOPOLOGY linear (ii)MOLECULE TYPE cDNA (iii) HYPOTHETICAL no (iv) ANTI-SENSE no (vi) ORIGINAL SOURCE (A) ORGANISM human (ix) FEATURE cDNA for Huntingtin-interacting protein (xi)SEQUENCE DESCRIPTION SEQ ID NO 1 ACAGCTGACA CCCTGCAAGG CCACCGGGAC CGCTTCATGG AGCAGTTTAC 50 AAAGTTGAAA GATCTGTTCT ACCGCTCCAG CAACCTGCAG TACTTCAAGC 100 GGGTCATTCA GATCCCCCAG CTGCCTGAGA ACCCACCCAA CTTCCTGCGA 150 GCCTCAGCCC TGTCAGAACA TATCAGCCCT GTGGTGGTGA TCCCTGCAGA 200 GGCCTCATCC CCCGACAGCG AGCCAGTCCT AGAGAAGGAT GACCTCATGG 250 ACATGGATGC CTCTCAGCAG AATTTATTTG ACAACAAGTT TGATGACNTC 300 TTTGGCAGTT CATCCAGCAG TGATCCCTTC AATTTCAACA GTCAAAATGG 350 TGTGAACAAG GATGAGAAGG ACCACTTAAT TGAGCGACTA TACAGAGAGA 400 TCAGTGGATT GAAGGCACAG CTAGAAAACA TGAAGACTGA GAGCCAGCGG 450 GTTGTGCTGC AGCTGAAGGG CCACGTCAGC GAGCTGGAAG CAGATCTGGC 500 CGAGCAGCAG CACCTGCGGC AGCAGGCGGC CGACGACTGT GAATTCCTGC 550 GGGCAGAACT GGACGAGCTC AGGNGGCAGC GGGAGGACAC CGAGAAGGCT 600 CAGCGGAGCC TGTCTGAGAT AGAAAGGAAA GCTCAAGCCA ATGAACAGCG 650 ATATAGCAAG CTAAAGGAGA AGTACAGCGA GCTGGTTCAG AACCACGCTG 700 ACCTGCTGCG GAAGAATGCA GAGGTGACCA AACAGGTGTC CATGGCCAGA 750 CAAGCCCAGG TAGATTTGGA ACGAGAGAAA AAAGAGCTGG AGGATTCGTT 800 GGAGCGCATC AGTGACCAGG GCCAGCGGAA GACTCAAGAA CAGCTGGAAG 850 TTCTAGAGAG CTTGAAGCAG GAACTTGGCA CAAGCCAACG GGAGCTTCAG 900 GTTCTGCAAG GCAGCCTGGA AACTTCTGCC CAGTCAGAAG CAAACTGGGC 950 AGCCGAGTTC GCCGAGCTAG AGAAGGAGCG GGACAGCCTG GTGAGTGGCG 1000 CAGCTCATAG GGAGGAGGAA TTATCTGCTC TTCGGAAAGA ACTGCAGGAC 1050 ACTCAGCTCA AACTGGCCAG CACAGAGGAA TCTATGTGCC AGCTTGCCAA 1100 AGACCAACGA AAAATGCTTC TGGTGGGGTC CAGGAAGGCT GCGGAGCAGG 1150 TGATACAAGA CGCG 1164 (2) INFORMATION FOR SEQ ID NO 2 (i) SEQUENCE CHARACTERISTICS (A) LENGTH 386 (B) TYPE protein (D) TOPOLOGY linear (ii)MOLECULE TYPE protein (iii) HYPOTHETICAL no (vi) ORIGINAL SOURCE (A) ORGANISM human (ix) FEATURE Huntingtin-interacting protein (xi) SEQUENCE DESCRIPTION SEQ ID N0.2 Thr Ala Asp Thr Leu Gin Gly His Arg Asp Arg Phe Met Glu Gin 1 5 10 15 Phe Thr Lys Leu Lys Asp Leu Phe Tyr Arg Ser Ser Asn Leu Gin 20 25 30 Tyr Phe Lys Arg Val lie Gin lie Pro Gin Leu Pro Glu Asn Pro 35 40 45 Pro Asn Phe Leu Arg Ala Ser Ala Leu Ser Glu His lie Ser Pro 50 55 60 Val Val Val He Pro Ala Glu Ala Ser Ser Pro Asp Ser Glu Pro 65 70 75 Val Leu Glu Lys Asp Asp Leu Met Asp Met Asp Ala Ser Gin Gin 80 85 90 Asn Leu Phe Asp Asn Lys Phe Asp Asp Phe Gly Ser Ser Ser Ser 95 100 105 Ser Asp Pro Phe Asn Phe Asn Ser Gin Asn Gly Val Asn Lys Asp 110 115 120 Glu Lys Asp His Leu He Glu Arg Leu Tyr Arg Glu He Ser Gly 125 130 135 Leu Lys Ala Gin Leu Glu Asn Met Lys Thr Glu Ser Gin Arg Val 140 145 150 Val Leu Gin Leu Lys Gly His Val Ser Glu Leu Glu Ala Asp Leu 155 160 165 Ala Glu Gin Gin His Leu Arg Gin Gin Ala Ala Asp Asp Cys Glu 170 175 180 Phe Leu Arg Ala Glu Leu Asp Glu Leu Arg Gin Arg Glu Asp Thr 185 190 195 Glu Lys Ala Gin Arg Ser Leu Ser Glu He Glu Arg Lys Ala Gin 200 205 210 Ala Asn Glu Gin Arg Tyr Ser Lys Leu Lys Glu Lys Tyr Ser Glu 215 220 225 Leu Val Gin Asn His Ala Asp Leu Leu Arg Lys Asn Ala Glu Val 230 235 240 Thr Lys Gin Val Ser Met Ala Arg Gin Ala Gin Val Asp Leu Glu 245 250 255 Arg Glu Lys Lys Glu Leu Glu Asp Ser Leu Glu Arg He Ser Asp 260 265 270 Gin Gly Gin Arg Lys Thr Gin Glu Gin Leu Glu Val Leu Glu Ser 275 280 285 Leu Lys Gin Glu Leu Gly Thr Ser Gin Arg Glu Leu Gin Val Leu 290 295 300 Gin Gly Ser Leu Glu Thr Ser Ala Gin Ser Glu Ala Asn Trp Ala 305 310 315 Ala Glu Phe Ala Glu Leu Glu Lys Glu Arg Asp Ser Leu Val Ser 320 325 330 Gly Ala Ala His Arg Glu Glu Glu Leu Ser Ala Leu Arg Lys Glu 335 340 345 Leu Gin Asp Thr Gin Leu Lys Leu Ala Ser Thr Glu Glu Ser Met 350 355 360 Cys Gin Leu Ala Lys Asp Gin Arg Lys Met Leu Leu Val Gly Ser 365 370 375 Arg Lys Ala Ala Glu Gin Val He Gin Asp Ala 380 385 386 (2) INFORMATION FOR SEQ ID NO 3 (i) SEQUENCE CHARACTERISTICS (A) LENGTH (B) TYPE nucleic acid (C) STRANDEDNESS single (D) TOPOLOGY linear (ii)MOLECULE TYPE cDNA (iii) HYPOTHETICAL no (iv) ANTI-SENSE no (vi) ORIGINAL SOURCE (A) ORGANISM human (ix) FEATURE cDNAfor Huntingtin-interacting protein (xi)SEQUENCE DESCRIPTION SEQ ID NO 3 ACCGATACCG AAGCGGGCTG TGTGCCCCTT CTCCACCCAG AGGAAATCAA 50 ACCCCAAAGC CATTATAACC ATGGATATGG TGAACCTCTT GGACGGAAAA 100 CTCATATTGA TGATTACAGC ACATGGGACA TAGTCAAGGC TACACAATAT 150 GGAATATATG AACGCTGTCG AGAATTGGTG GAAGCAGGTT ATGATGTACG 200 GCAACCGGAC AAAGAAAATG TTACCCTCCT CCATTGGGCT GCCATCAATA 250 ACAGAATAGA TTTAGTCAAA TACTATATTT CGAAAGGTGC TATTGTGGAT 300 CAACTTGGAG GGGACCTGAA TTCAACTCCA TTGCACTGGG ACACAAGACA 350 AGGCCATCTA TCCATGGTTG TGCAACTAAT GAAATATGGT GCAGATCCTT 400 CATTAATTGA TGGAGAAGGA TGTAGCTGTA TTCATCTGGC TGCTCAGTTC 450 GGACATACCT CAATTGTTGC TTATCTCATA GCAAAAGGAC AGGATGTG 498 (2) INFORMATION FOR SEQ ID NO 4 (i) SEQUENCE CHARACTERISTICS (A) LENGTH 154 (B) TYPE protein (D) TOPOLOGY linear (ii)MOLECULE TYPE protein (iii) HYPOTHETICAL no (vi) ORIGINAL SOURCE (A) ORGANISM human (ix) FEATURE Huntingtin-interacting protein (xi) SEQUENCE DESCRIPTION SEQ ID NO 4 Thr Asp Thr Glu Ala Gly Cys Val Pro Leu Leu His Pro Glu Glu 1 5 10 15 He Lys Pro Gin Ser His Tyr Asn His Gly Tyr Gly Glu Pro Leu 20 25 30 Gly Arg Lys Thr His He Asp Asp Tyr Ser Thr Trp Asp He Val 35 40 45 Lys Ala Thr Gin Tyr Gly He Tyr Glu Arg Cys Arg Glu Leu Val 50 55 60 Glu Ala Gly Tyr Asp Val Arg Gin Pro Asp Lys Glu Asn Val Thr 65 70 75 Leu Leu His Trp Ala Ala He Asn Asn Arg He Asp Leu Val Lys 80 85 90 Tyr Tyr He Ser Lys Gly Ala He Val Asp Gin Leu Gly Gly Asp 95 100 105 Leu Asn Ser Thr Pro Leu His Trp Asp Thr Arg Gin Gly His Leu 110 115 120 Ser Met Val Val Gin Leu Met Lys Tyr Gly Ala Asp Pro Ser Leu 125 130 135 He Asp Gly Glu Gly Cys Ser Cys He His Leu Ala Ala Gin Phe 140 145 150 Gly His Thr Ser 154 (2) INFORMATION FOR SEQ ID NO 5 (i) SEQUENCE CHARACTERISTICS (A) LENGTH 4846 (B) TYPE nucleic acid (C) STRANDEDNESS single (D) TOPOLOGY linear (iι)MOLECULE TYPE cDNA (iii) HYPOTHETICAL no (iv) ANTI-SENSE no (vi) ORIGINAL SOURCE (A) ORGANISM human (ix) FEATURE cDNA for Huntingtin-interacting protein (xi)SEQUENCEDESCRIPTION SEQ IDNO 5 CAGTGTACGG TTGATCATAT AACGCCGCGG GCGGGGATTG GTTTATATAT 50 CGCAAATTGA TNTAGGGGGG GGGGGATGGN CAGAGATTTC GCTTCATTAG 100 GCCATTATAA GCAGGAAGGG TTTCAAGGAA AAAAACCCAG AAAGTGCATA 150 TTGCACCCAC CATGAGAAAG GGGCAACAGA CCTTNTGTTN TGTTNTCAAC 200 CGCCTGCTTC TGTTTTAGCA ACGCAGTGTT TTGGTGGAAG TTGTGCCATG 250 TGTTCCACAA ANTCTTCCGA GATGGACACC CGAACGTCCT GAAGGACTTT 300 GTGAGATACA GAAATGAATT GAGTGACATG AGCAGGATGT GGGGCCACCT 350 GAGCGAGGGG TATGGCCAGC TGTGCAGCAT CTACCTGAAA CTGCTAAGAA 400 CCAAGATGGA GTACCACACC AAAAATCCCA GGTTCCCAGG CAACCTGCAG 450 ATGAGTGACC GCCAGCTGGA CGAGGCTGGA GAAAGTGACG TGAACAACTT 500 TTTCCAGTTA ACAGTGGAGA TGTTTGACTA CCTGGAGTGT GAACTCAACC 550 TCTTCCAAAC AGTATTCAAC TCCCTGGACA TGTCCCGCTC TGTGTCCGTG 600 ACGGCAGCAG GGCAGTGCCG CCTCGCCCCG CTGATCCAGG TCATCTTGGA 650 CTGCAGCCAC CTTTATGACT ACACTGTCAA GCTTCTCTTC AAACTCCACT 700 CCTGCCTCCC AGCTGACACC CTGCAAGGCC ACCGGGACCG CTTCATGGAG 750 CAGTTTACAA AGTTGAAAGA TCTGTTCTAC CGCTCCAGCA ACCTGCAGTA 800 CTTCAAGCGG CTCATTCAGA TCCCCCAGCT GCCTGAGAAC CCACCCAACT 850 TCCTGCGAGC CTCAGCCCTG TCAGAACATA TCAGCCCTGT GGTGGTGATC 900 CCTGCAGAGG CCTCATCCCC CGACAGCGAG CCAGTCCTAG AGAAGGATGA 950 CCTCATGGAC ATGGATGCCT CTCAGCAGAA TTTATTTGAC AACAAGTTTG 1000 ATGACATCTT TGGCAGTTCA TTCAGCAGTG ATCCCTTCAA TTTCAACAGT 1050 CAAAATGGTG TGAACAAGGA TGAGAAGGAC CACTTAATTG AGCGACTATA 1100 CAGAGAGATC AGTGGATTGA AGGCACAGCT AGAAAACATG AAGACTGAGA 1150 GCCAGCGGGT TGTGCTGCAG CTGAAGGGCC ACGTCAGCGA GCTGGAAGCA 1200 GATCTGGCCG AGCAGCAGCA CCTGCGGCAG CAGGCGGCCG ACGACTGTGA 1250 ATTCCTGCGG GCAGAACTGG ACGAGCTCAG GAGGCAGCGG GAGGACACCG 1300 AGAAGGCTCA GCGGAGCCTG TCTGAGATAG AAAGGAAAGC TCAAGCCAAT 1350 GAACAGCGAT ATAGCAAGCT AAAGGAGAAG TACAGCGAGC TGGTTCAGAA 1400 CCACGCTGAC CTGCTGCGGA AGAATGCAGA GGTGACCAAA CAGGTGTCCA 1450 TGGCCAGACA AGCCCAGGTA GATTTGGAAC GAGAGAAAAA AGAGCTGGAG 1500 GATTCGTTGG AGCGCATCAG TGACCAGGGC CAGCGGAAGA CTCAAGAACA 1550 GCTGGAAGTT CTAGAGAGCT TGAAGCAGGA ACTTGGCACA AGCCAACGGG 1600 AGCTTCAGGT TCTGCAAGGC AGCCTGGAAA CTTCTGCCCA GTCAGAAGCA 1650 AACTGGGCAG CCGAGTTCGC CGAGCTAGAG AAGGAGCGGG ACAGCCTGGT 1700 GAGTGGCGCA GCTCATAGGG AGGAGGAATT ATCTGCTCTT CGGAAAGAAC 1750 TGCAGGACAC TCAGCTCAAA CTGGCCAGCA CAGAGGAATC TATGTGCCAG 1800 CTTGCCAAAG ACCAACGAAA AATGCTTCTG GTGGGGTCCA GGAAGGCTGC 1850 GGAGCAGGTG ATACAAGACG CCCTGAACCA GCTTGAAGAA CCTCCTCTCA 1900 TCAGCTGCGC TGGGTCTGCA GATCACCTCC TCTCCACGGT CACATCCATT 1950 TCCAGCTGCA TCGAGCAACT GGAGAAAAGC TGGAGCCAGT ATCTGGCCTG 2000 CCCAGAAGAC ATCAGTGGAC TTCTCCATTC CATAACCCTG CTGGCCCACT 2050 TGACCAGCGA CGCCATTGCT CATGGTGCCA CCACCTGCCT CAGAGCCCCA 2100 CCTGAGCCTG CCGACTCACT GACCGAGGCC TGTAAGCAGT ATGGCAGGGA 2150 AACCCTCGCC TACCTGGCCT CCCTGGAGGA AGAGGGAAGC CTTGAGAATG 2200 CCGACAGCAC AGCCATGAGG AACTGCCTGA GCAAGATCAA GGCCATCGGC 2250 GAGGAGCTCC TGCCCAGGGG ACTGGACATC AAGCAGGAGG AGCTGGGGGA 2300 CCTGGTGGAC AAGGAGATGG CGGCCACTTC AGCTGCTATT GAAACTTGCA 2350 CGGCCAGAAT AGAGGAGATG CTCAGCAAAT CCCGAGCAGG AGACACAGGA 2400 GTCAAATTGG AGGTGAATGA AAGGATCCTT CGTTGCTGTA CCAGCCI'CAT 2450 GCAAGCTATT CAGGTGCTCA TCGTGGCCTC TAAGGACCTC CAGAGAGAGA 2500 TTGTGGAGAG CGGCAGGGGT ACAGCATCCC CTAAAGAGTT TTATGCCAAG 2550 AACTCTCGAT GGACAGAAGG ACTTATCTCA GCCTCCAAGG CTGTGGGCTG 2600 GGGAGCCACT GTCATGGTGG ATGCAGCTGA TCTGGTGGTA CAAGGCAGAG 2650 GGAAATTTGA GGAGCTAATG GTGTGTTCTC ATGAAATTGC TGCTAGCACA 2700 GCCCAGCTTG TGGCTGCATC CAAGGTGAAA GCTGATAAGG ACAGCCCCAA 2750 CCTAGCCCAG CTGCAGCAGG CCTCTCGGGG AGTGAACCAG GCCACTGCCG 2800 GCGTTGTGGC CTCAACCATT TCCGGCAAAT CACAGATCGA AGAGACAGAC 2850 AACATGGACT TCTCAAGCAT GACGCTGACA CAGATCAAAC GCCAAGAGAT 2900 GGATTCTCAG GTTAGGGTGC TAGAGCTAGA AAATGAATTG CAGAAGGAGC 2950 GTCAAAAACT GGGAGAGCTT CGGAAAAAGC ACTACGAGCT TGCTGGTGTT 3000 GCTGAGGGCT GGGAAGAAGG AACAGAGGCA TCTCCACCTA CACTGCAAGA 3050 AGTGGTAACC GAAAAAGAAT AGAGCCAAAC CAACACCCCA TATGTCAGTG 3100 TAAATCCTTG TTACCTATCT CGTGTGTGTT ATTTCCCCAG CCACAGGCCA 3150 AATCCTTGGA GTCCCAGGGG CAGCCACACC ACTGCCATTA CCCAGTGCCG 3200 AGGACATGCA TGACACTTCC CAAAGATCCC TCCATAGCGA CACCCTTTCT 3250 GTTTGGACCC ATGGTCATCT CTGTTCTTTT CCCGCCTCCC TAGTTAGCAT 3300 CCAGGCTGGC CAGTGCTGCC CATGAGCAAG CCTAGGTACG AAGAGGGGTG 3350 GTGGGGGGCA GGGCCACTCA ACAGAGAGGA CCAACATCCA GTCCTGCTGA 3400 CTATTTGACC CCCACAACAA TGGGTATCCT TAATAGAGGA GCTGCTTGTT 3450 GTTTGTTGAC AGCTTGGAAA GGGAAGATCT TATGCCTTTT CTTTTCTGTT 3500 TTCTTCTCAG TCTTTTCAGT TTCATCATTT GCACAAACTT GTGAGCATCA 3550 GAGGGCTGAT GGATTCCAAA CCAGGACACT ACCCTGAGAT CTGCACAGTC 3600 AGAAGGACGG CAGGAGTGTC CTGGCTGTGA ATGCCAAAGC CATTCTCCCC 3650 CTCTTTGGGC AGTGCCATGG ATTTCCACTG CTTCTTATGG TGGTTGGTTG 3700 GGTTTTTTGG TTTTGTTTTT TTTTTTTAAG TTTCACTCAC ATAGCCAACT 3750 CTCCCAAAGG GCACACCCCT GGGGCTGAGT CTCCAGGGCC CCCCAACTGT 3800 GGTAGCTCCA GCGATGGTGC TGCCCAGGCC TCTCGGTGCT CCATCTCCGC 3850 CTCCACACTG ACCAAGTGCT GGCCCACCCA GTCCATGCTC CAGGGTCAGG 3900 CGGAGCTGCT GAGTGACAGC TTTCCTCAAA AAGCAGAAGG AGAGTGAGTG 4000 CCTTTCCCTC CTAAAGCTGA ATCCCGGCGG AAAGCCTCTG TCCGCCTTTA 4050 CAAGGGAGAA GACAACAGAA AGAGGGACAA GAGGGTTCAC ACAGCCCAGT 4100 TCCCGTGACG AGGCTCAAAA ACTTGATCAC ATGCTTGAAT GGAGCTGGTG 4150 AGATCAACAA CACTACTTCC CTGCCGGAAT GAACTGTCCG TGAATGGTCT 4200 CTGTCAAGCG GGCCGTCTCC CTTGGCCCAG AGACGGAGTG TGGGAGTGAT 4250 TCCCAACTCC TTTCTGCAGA CGTCTGCCTT GGCATCCTCT TGAATAGGAA 4300 GATCGTTCCA CTTTCTACGC AATTGACAAA CCCGGAAGAT CAGATGCAAT 4350 TGCTCCCATC AGGGAAGAAC CCTATACTTG GTTTGCTACC CTTAGTATTT 4400 ATTACTAACC TCCCTTAAGC AGCAACAGCC TACAAAGAGA TGCTTGGAGC 4450 AATCAGAACT TCAGGTGTGA CTCTAGCAAA GCTCATCTTT CTGCCCGGCT 4500 ACATCAGCCT TCAAGAATCA GAAGAAAGCC AAGGTGCTGG ACTGTTACTG 4550 ACTTGGATCC CAAAGCAAGG AGATCATTTG GAGCTCTTGG GTCAGAGAAA 4600 ATGAGAAAGG ACAGAGCCAG CGGCTCCAAC TCCTTTCAGC CACATGCCCC 4650 AGGCTCTCGC TGCCCTGTGG ACAGGATGAG GACAGAGGGC ACATGAACAG 4700 CTTGCCAGGG ATGGGCAGCC CAACAGCACT TTTCCTCTTC TAGATGGACC 4750 CCAGCATTTA AGTGACCTTC TGATCTTGGG AAAACAGCGT CTTCCTTCTT 4800 TATCTATAGC AACTCATTGG TGGTAGCCAT CAAGCACTTC GGAATT 4846 (2) INFORMATION FOR SEQ ID NO 6 (i) SEQUENCE CHARACTERISTICS (A) LENGTH. 924 (B) TYPE protein (D) TOPOLOGY linear (ii)MOLECULE TYPE, protein (iii) HYPOTHETICAL, no (vi) ORIGINAL SOURCE (A) ORGANISM human (ix) FEATURE Huntingtin-interacting protein (xi) SEQUENCE DESCRIPTION SEQ ID NO 6 Met Ser Arg Met Trp Gly His Leu Ser Glu Gly Tyr Gly Gin Leu 1 5 10 * 15 Cys Ser He Tyr Leu Lys Leu Leu Arg Thr Lys Met Glu Tyr His 20 25 30 Thr Lys Asn Pro Arg Phe Pro Gly Asn Leu Gin Met Ser Asp Arg 35 40 45 Gin Leu Asp Glu Ala Gly Glu Ser Asp Val Asn Asn Phe Phe Gin 50 55 60 Leu Thr Val Glu Met Phe Asp Tyr Leu Glu Cys Glu Leu Asn Leu 65 70 75 Phe Gin Thr Val Phe Asn Ser Leu Asp Met Ser Arg Ser Val Ser 80 85 90 Val Thr Ala Ala Gly Gin Cys Arg Leu Ala Pro Leu He Gin Val 95 100 105 He Leu Asp Cys Ser His Leu Tyr Asp Tyr Thr Val Lys Leu Leu 110 115 120 Phe Lys Leu His Ser Cys Leu Pro Ala Asp Thr Leu Gin Gly His 125 130 135 Arg Asp Arg Phe Met Glu Gin Phe Thr Lys Leu Lys Asp Leu Phe 140 145 150 Tyr Arg Ser Ser Asn Leu Gin Tyr Phe Lys Arg Leu He Gin He 155 160 165 Pro Gin Leu Pro Glu Asn Pro Pro Asn Phe Leu Arg Ala Ser Ala 170 175 180 Leu Ser Glu His He Ser Pro Val Val Val He Pro Ala Glu Ala 185 190 195 Ser Ser Pro Asp Ser Glu Pro Val Leu Glu Lys Asp Asp Leu Met 200 205 210 Asp Met Asp Ala Ser Gin Gin Asn Leu Phe Asp Asn Lys Phe Asp 215 220 225 Asp He Phe Gly Ser Ser Phe Ser Ser Asp Pro Phe Asn Phe Asn 230 235 240 Ser Gin Asn Gly Val Asn Lys Asp Glu Lys Asp His Leu He Glu 245 250 255 Arg Leu Tyr Arg Glu He Ser Gly Leu Lys Ala Gin Leu Glu Asn 260 265 270 Met Lys Thr Glu Ser Gin Arg Val Val Leu Gin Leu Lys Gly His 275 280 285 Val Ser Glu Leu Glu Ala Asp Leu Ala Glu Gin Gin His Leu Arg 290 295 300 Gin Gin Ala Ala Asp Asp Cys Glu Phe Leu Arg Ala Glu Leu Asp 305 310 315 Glu Leu Arg Arg Gin Arg Glu Asp Thr Glu Lys Ala Gin Arg Ser 320 325 330 Leu Ser Glu He Glu Arg Lys Ala Gin Ala Asn Glu Gin Arg Tyr 335 340 345 Ser Lys Leu Lys Glu Lys Tyr Ser Glu Leu Val Gin Asn His Ala 350 355 360 Asp Leu Leu Arg Lys Asn Ala Glu Val Thr Lys Gin Val Ser Met 365 370 375 Ala Arg Gin Ala Gin Val Asp Leu Glu Arg Glu Lys Lys Glu Leu 380 385 390 Glu Asp Ser Leu Glu Arg He Ser Asp Gin Gly Gin Arg Lys Thr 395 400 405 Gin Glu Gin Leu Glu Val Leu Glu Ser Leu Lys Gin Glu Leu Gly 410 415 420 Thr Ser Gin Arg Glu Leu Gin Val Leu Gin Gly Ser Leu Glu Thr 425 430 435 Ser Ala Gin Ser Glu Ala Asn Trp Ala Ala Glu Phe Ala Glu Leu 440 445 450 Glu Lys Glu Arg Asp Ser Leu Val Ser Gly Ala Ala His Arg Glu 455 460 465 Glu Glu Leu Ser Ala Leu Arg Lys Glu Leu Gin Asp Thr Gin Leu 470 475 480 Lys Leu Ala Ser Thr Glu Glu Ser Met Cys Gin Leu Ala Lys Asp 485 490 495 Gin Arg Lys Met Leu Leu Val Gly Ser Arg Lys Ala Ala Glu Gin 500 505 510 Val He Gin Asp Ala Leu Asn Gin Leu Glu Glu Pro Pro Leu He 515 520 525 Ser Cys Ala Gly Ser Ala Asp His Leu Leu Ser Thr Val Thr Ser 530 535 540 He Ser Ser Cys He Glu Gin Leu Glu Lys Ser Trp Ser Gin Tyr 545 550 555 Leu Ala Cys Pro Glu Asp He Ser Gly Leu Leu His Ser He Thr 560 565 570 Leu Leu Ala His Leu Thr Ser Asp Ala He Ala His Gly Ala Thr 575 580 585 Thr Cys Leu Arg Ala Pro Pro Glu Pro Ala Asp Ser Leu Thr Glu 590 595 600 Ala Cys Lys Gin Tyr Gly Arg Glu Thr Leu Ala Tyr Leu Ala Ser 605 610 615 Leu Glu Glu Glu Gly Ser Leu Glu Asn Ala Asp Ser Thr Ala Met 620 625 630 Arg Asn Cys Leu Ser Lys He Lys Ala He Gly Glu Glu Leu Leu 635 640 645 Pro Arg Gly Leu Asp He Lys Gin Glu Glu Leu Gly Asp Leu Val 650 655 660 Asp Lys Glu Met Ala Ala Thr Ser Ala Ala He Glu Thr Cys Thr 665 670 675 Ala Arg He Glu Glu Met Leu Ser Lys Ser Arg Ala Gly Asp Thr 680 685 690 Gly Val Lys Leu Glu Val Asn Glu Arg He Leu Arg Cys Cys Thr 695 700 705 Ser Leu Met Gin Ala He Gin Val Leu He Val Ala Ser Lys Asp 710 715 720 Leu Gin Arg Glu He Val Glu Ser Gly Arg Gly Thr Ala Ser Pro 725 730 735 Lys Glu Phe Tyr Ala Lys Asn Ser Arg Trp Thr Glu Gly Leu He 740 745 750 Ser Ala Ser Lys Ala Val Gly Trp Gly Ala Thr Val Met Val Asp 765 770 775 Ala Ala Asp Leu Val Val Gin Gly Arg Gly Lys Phe Glu Glu Leu 780 785 790 Met Val Cys Ser His Glu He Ala Ala Ser Thr Ala Gin Leu Val 795 800 805 Ala Ala Ser Lys Val Lys Ala Asp Lys Asp Ser Pro Asn Leu Ala 810 815 820 Gin Leu Gin Gin Ala Ser Arg Gly Val Asn Gin Ala Thr Ala Gly 825 830 835 Val Val Ala Ser Thr He Ser Gly Lys Ser Gin He Glu Glu Thr 840 845 850 Asp Asn Met Asp Phe Ser Ser Met Thr Leu Thr Gin He Lys Arg 855 860 865 Gin Glu Met Asp Ser Gin Val Arg Val Leu Glu Leu Glu Asn Glu 870 875 880 Leu Gin Lys Glu Arg Gin Lys Leu Gly Glu Leu Arg Lys Lys His 885 890 895 Tyr Glu Leu Ala Gly Val Ala Glu Gly Trp Glu Glu Gly Thr Glu 900 905 910 Ala Ser Pro Pro Thr Leu Gin Glu Val Val Thr Glu Lys Glu 915 920 924

Claims: CLAIMS 1 A cDNA molecule comprising the sequence given by Seq ID No 1 2 A cDNA molecule comprising the sequence given by Seq ID No 5 3 A polypeptide comprising the sequence given by Seq ID No 2 4 A polypeptide comprising the sequence given by Seq. ID No 6 5 A chimeric gene or plasmid comprising at least nucleotides 314 to 1955 of the Huntington's Disease gene and an activating or DNA binding domain suitable for use in a yeast multi-hybrid assay 6 The chimeric gene or plasmid according to claim 5, wherein the Huntington's Disease gene encodes a polyglutamine tract having a length of 35 or fewer residues 7 The chimeric gene or plasmid according to claim 5, wherein the Huntington's Disease gene encodes a polyglutamine tract having a length of 36 or more residues 8 A method for ameliorating the effects of Huntington's disease in a patient expressing Huntingtin protein with an expanded CAG repeat region, comprising the step of increasing the amount of an expressed HD-interacting polypeptide in the brain of the patient, wherein the expressed HD-interacting polypeptide interacts less well with expanded Huntingtin than with Huntingtin having a CAG repeat region containing 15 to 35 repeats and facilitates the incorporation of Huntingtin into brain cell membranes 9 The method according to claim 8, wherein the expressed HD- interacting polypeptide comprises the sequence given by Seq ID No 2 10 An antibody which binds to a polypeptide having the sequence given by Seq ID No 2 1 1 The antibody of claim 10. wherein the antibody binds to amino acids 76-91 of the polypeptide having the sequence shown in Seq. ID No. 2. 12. An expression vector for expression of a gene in a mammalian host comprising a region encoding an HD-interacting polypeptide, wherein the HD-interacting polypeptide interacts less well with expanded Huntingtin than with Huntingtin having a CAG repeat region containing 1 5 to 35 repeats and facilitates the incorporation of Huntingtin into brain cell membranes 13. An expression vector for expression of a gene in a mammalian host comprising a region that is the same as or complementary to Seq. ID NO. 1. 14. An expression vector for expression of a gene in a mammalian host comprising a region that is the same as or complementary to Seq. ID NO. 5. 15. The expression vector according to claims of claims 12-14, further comprising a region encoding Huntingtin having a polyglutamine tract of 35 or fewer. 16 An oligonucleotide probe having a length of from 15-40 bases which specifically and selectively hybridizes with the cDNA given by Seq ID No 1 or a sequence complementary thereto

Inventors: HAYDEN MICHAEL R, KALCHMAN MICHAEL, HAYDEN, MICHAEL, R., KALCHMAN, MICHAEL
Applicants: HAYDEN MICHAEL R, KALCHMAN MICHAEL, UNIV BRITISH COLUMBIA, HAYDEN, MICHAEL, R., KALCHMAN, MICHAEL, THE UNIVERSITY OF BRITISH COLUMBIA
Application year: 1996
Main classification: A61K38
IPC codes: A61K38, C07K14, C07K16