CA2285690A1 - Gene necessary for striatal function and uses thereof - Google Patents

Abstract

The present invention provides nucleotide sequences that identify and encode a novel gene (DHD) that is normally highly expressed in mammalian striatum, nucleus accumbens and olfactory tubercule. Decreases in expression of this gene accompany the development of CAG
repeat disorders such as Huntington's disease. The present invention also provides for antisense molecules to the nucleotides, methods for measuring the expression of the sequence, expression vectors for the sequence, and genetically engineered host cells for the expression of DHD. This novel sequence is particularly useful for screening candidate compounds for their ability to prevent neurodegeneration in various neurological disorders.

Description

Gene Necessar~for Striatal Function and Uses Thereof FIELD OF THE INVENTION
The present invention relate;; to polynucleotides expressed in the brain and which are involved in neurological disorders. Wore particularly, the present invention relates to polynucleotides expressed in the striatum, nucleus accumbens and olfactory tubercule, and which are down-regulated in the course of CAG repeat disorders, such as Huntington's disease.
The present invention also describes variants and derivatives of these polynucleotides;
processes for making these polynucleotides, and their agonists and antagonists, and uses of these polynucleotides, variants, derivatives, agonists and antagonists.
BACKGROUND OF THE INVENTION
Very few if any ei~ective treatments exist for neurological disorders characterized by progressive cell loss, known as neurodegenerative diseases, as well as those involving acute cell loss, such as stroke and trauma.
Huntington's disease (HD) is an inherited neurological disorder that is transmitted in autosomal dominant fashion. HD results from genetically programmed degeneration of neurons in certain areas of t',he brain. Huntington's disease is caused by a mutation of the gene IT IS that codes for the protein huntingtin. The huntingtin gene contains a polymorphic stretch of repeated CAG trinucleotides that encode a polyglutamine tract within huntingtin. If this tract exceeds 35 in number, Huntington's disease results. Huntington's disease is only one of a number of neurological diseases which are characterised by these polyglutamine repeats (Ross, 1997). Schizophrenia, Alzheimer's disease, stroke, trauma, and Parkinson's disease also affect the basal ganglia.
Huntingtin has no sequence similarity to known proteins (Group THDCR, 1993;
Sisodia, 1998). The function of the normal or mutated HD form of huntingtin has not been defined by the prior art. It is evident, however, that the expression of the HD form of huntingtin leads to progressive and selective neuronal loss. It has been demonstrated that the GABA- and enkephalin-containing medium spiny projection neurons of the caudate-putamen eventually die as a result of HD (Richfield et al., 1994). Patients with minimal cell loss, however, still present with motor and cognitive symptoms suggesting that neuronal dysfunction, and not simply cell loss, contribute to the symptoms of HD. The motor symptoms of HD
include the development of chorea, dysl:onia, bradykinesia and tremors (Young et al., 1986). Voluntary 1 S movements may also be affected such that there may be disturbances in speech (Ludlow et al., 1987) and degradation of fine motor co-ordination (Young et al., 1986). In addition to motor decline, emotional disturbances and cognitive loss are also evident during the progression of HD (Came et al., 1978).
Despite the fact that huntin~;tin is ubiquitously expressed, ITD specifically affects cells of the basal ganglia, structures deep within the brain that have a number of important functions, including co-ordinating movement. The basal ganglia includes the caudate nucleus, the putamen, the nucleus accumbens and the olfactory tubercule. HD also affects the brain's outer surface, or cortex, which controls thought, perception, and memory. The mechanism by which only a small group of neurons in the striatum and cortex are rendered vulnerable to this ubiquitously expressed mutant protein is not known. There are no effective treatments for Huntington's disease.
Huntington's disease is widf;ly believed to be a gain-of function disorder but neither the normal function nor the gained function of huntingtin is known. Because the function for huntingtin is not known, there is little insight into the disease process. It was believed that huntingtin was related to neuronal intranuclear inclusions (NII). However, recent results have cast doubt on our understanding of the role of the NII in Huntington's disease (Saudou et al., 1998) or in other CAG repeat disorders (Klement et al., 1998; see also commentary by Sisodia, 1998).
The development of a mouse carrying the S' end of the human Huntington's disease gene (the promoter and first exon; Mangiarini et al., 1996) was an important step in the development of the tools that will allow us to understand the function (and gain-of function) associated with huntingtin. R6/2 mice exhibit a rapidly progressing neurological phenotype with onset at about 8 weeks. This phenotype includes a movement disorder characterised by shuddering, resting tremor, epileptic sei:~ures and stereotyped behaviour. These symptoms suggest that the function of the basal ganglia is affected by the expression of the human exon 1 transgene prior to neuronal cell death. By 12 weeks the affected mice have significantly reduced brain weights and they die by about 13 weeks of age. Neuronal intranuclear inclusions (NII) develop at about 4 weeks (Davies et al., 1997). As is observed in human Huntington's disease patient, the R6/2 mice show changes in neuronal receptors (Cha et al., 1998). The present inventors have also demonstrated that changes in the expression of DARPP-32 and cannabinoid receptors change over time in HD mice; such changes have also been observed in human Huntington's disease patients (unpublished results). The loss of the cannabinoid receptor is one of the earliest documented changes that occur prior to neuronal degeneration in human HD patients. The R6/2 model, therefore, mimics the early phases of HD; a point in disease development where intervention would be most appropriate.
SUMMARY OF THE INVENTION
The present invention provides nucleotide sequences that identify and encode a novel gene (DHD) that is normally highly expressed in mammalian striatum, nucleus accumbens and olfactory tubercule. The invention teaches an isolated polynucleotide segment, comprising a polynucleotide sequence selected from the group consisting of (a) a sequence comprising SEQ ID NO:1; (b) a sequence comprising SEQ ID N0:2; (c) a sequence having nucleotides 1140 to 3235 of SEQ ID NO:1; (d) a sequence which is at least 80% homologous with a sequence of (a), (b) or (c); ~(e) variants of (a), (b), (c) or (d), and; (~ a sequence which hybridizes to (a), (b), (c) or (d) under stringent conditions. In a preferred embodiment, the isolated polynucleotide segment is RNA. The invention also teaches an isolated polynucleotide segment, which retains substantially the same biological function or activity as the polynucleotide encoded by the polynucleotide sequence.
Further preferred embodiments of the invention are polynucleotides that are at least 70%
identical over their entire length to a polynucleotide encoding DHD
polypeptide or S

polynucleotide, and polynucleotides which are complementary to such polynucleotides.
Alternatively, most highly preferred are polynucleotides that comprise a region that is at least 80% identical over their entire length to a polynucleotide encoding DHD of SEQ
ID NO. and polynucleotides complementary thereto. In this regard, polynucleotides at least 90% identical over their entire length to the same are particularly preferred, and among these particularly preferred polynucleotides, those with at least 95% are especially preferred.
Furthermore, those with at least 97% are highly preferred among those with at least 95%, and among these those with at least 98% and at least 99% are particularly highly preferred, with at least 99% being the more preferred.
The invention also provides for a vector with the isolated polynucleotide segment, and an isolated host cell comprising; the vector. The invention also teaches a process for producing a polypeptide of a polynucleotide sequence comprising the step of culturing the host cell under conditions sufFlcient for the production of said polypeptide.
The invention further providles an isolated polynucleotide fragment of the polynucleotide sequence, wherein the polynucleotide sequence is selected from the group consisting o~ (a) a sequence having at least 1 S sequential bases of nucleotides 1140 to 323 5 of SEQ ID NO: 1;
(b) a sequence having at least 30 sequential bases of nucleotides 1140 to 3235 of SEQ ID NO:
1;
(c) a sequence having at least 50 sequential bases of SEQ ID NO:1 or SEQ ID
NO: 2; (d) a sequence which is at least 90% homologous with a sequence of (a), (b) or (c);
(e) variants of (a), (b), (c) or (d), and; (fj a. sequence which hybridizes to (a), (b), (c) or (d) under stringent conditions.

The invention also teaches a method for identifying a compound which inhibits or promotes the activity of a polynucleotide segment of claim 1, comprising the steps o~
(a) selecting a control animal having said segment and a test animal having said segment; (b) treating said test animal using a compound; and (c) determining the relative quantity of RNA
corresponding to said segment, as between said animals. In a preferred embodiment, the animal is a mammal, preferably a mouse, and preferably a transgenic mouse.
The invention also teaches a method for identifying a compound which inhibits or promotes the activity of a polynucleotide segment of claim 1, comprising the steps of (a) selecting a host cell of claim 4; (b) cloning said host cell and separating said clones into a test group and a control group; (c) treating said test group using a compound; and (c) determining the relative quantity of RNA corresponding to said segment, as between said test group and said control group.
The invention further teaches a method for diagnosing the presence of or the predisposition for a CAG repeat disorder, said method comprising determining the level of expression of RNA corresponding to the segments of claim 1 in an individual relative to a predetermined control level of expression, wherein a decreased expression of said RNA as compared to said control is indicative of a CAG repeat disorder. Preferably, the expression is measured by in situ hybridization, fluorescent in situ hybridization, polymerase chain reaction, or DNA
fingerprinting technique. In a preferred embodiment, the CAG repeat disorder is Huntington's disease.

BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 A is a portion of an autoradiogram of the differential display reaction identifying DHD
in mouse brain mRNA.
FIB. 1B is a northern blot confirming that DHD has a lower steady-state level of expression in the striatum of transgenic HD mice.
FIG. 2 is a nucleotide sequence of the differential display cDNA fragment pDHD.
FIG. 3A shows the in situ hybridization of probe 1 to coronal and saggital brain sections of 10 week-old wild-type and HD mice.
FIG. 3B shows the in situ hybridization corresponding to spatial and temporal expression of DHD in brain sections of wild-type and HD mice over the period of time that the HD mice develop abnormal movements and postures.
FIG. 3C shows the in situ hybridization corresponding to expression of DHD in brain sections of one day old wild-type and HD mice.
FIG. 3D shows the in situ hybridization corresponding to distribution of the mRNA of DHD
in mouse striatal neurons.

FIG. 4 is the in situ hybridi~:ation corresponding to mRNA distribution of the rat homologue of DHD in rat brain tissue.
FIG. S shows a Southern blot analysis of DNA from wild-type and transgenic HD
mice hybridized to the pDHD cDNA probe.
FIG. 6 is a nucleotide sequemce of cDHD-1, and corresponds to SEQ ID NO. 1.
FIG. 7 is a restriction map of cDHD-1.
FIG. 8 is a nucleotide segue-nce of cDHD-2, and corresponds to SEQ ID NO. 2.
FIG. 9 is a restriction map of cDHD-2.
1 S FIG. 10 is a schematic diagram showing the alignment of cDHD-1 and -2 and the regions that are identical and unique between the two clones.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION
The following illustrative e~;planations are provided to facilitate understanding of certain terms used frequently herein. The. explanations are provided as a convenience and are not limitative of the invention.
"Host cell" is a cell which h;as been transformed or transfected, or is capable of transformation or transfection by an exogenous polynucleotide sequence.
"Identity", "similarity" or "homologous", as used in the art, are relationships between two or more polynucleotide sequences, as determined by comparing the sequences. In the art, identity also means the degree of sequence relatedness between polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences.
Both identity and similarity can be readily calculated (Lesk, A. M., 1988; Smith, D. W., 1993; Griffin, A.
M., and Grii~rn, H. G., 1994; von Heinje, G., 1987; and Gribskov, M. and Devereux, J., 1991). While there exist a number of methods to measure identity and similarity between two polynucleotide sequences, both terms are well known to skilled artisans (von Heinje, G., 1987;
Gribskov, M. and Devereux, 1991; and Carillo, H., and Lipman, D., 1988).
Methods commonly employed to detc;rmine identity or similarity between sequences include, but are not limited to those disclosed in Carillo, H., and Lipman, D. (1988). Methods to determine identity and similarity are codified in computer programs. Preferred computer program methods to determine identity and similarity between two sequences include, but are not limited to, GCG program package (Devereux, J., et al., 1984), BLASTP, BLASTN, and FASTA (Atschul, S. F. et al.., 1990).
"Isolated" means altered "by the hand of man" from its natural state; i.e., that, if it occurs in nature, it has been changed or removed from its original environment, or both.
For example, a naturally occurnng polynuc:leotide naturally present in a living organism in its natural state is not "isolated," but the same polynucleotide separated from coexisting materials of its natural state is "isolated", as the term is employed herein. As part of or following isolation, such polynucleotides can be joined to other polynucleotides, such as DNA, for mutagenesis, to form fusion proteins, and for propagation or expression in a host, for instance. The isolated polynucleotides, alone or joined to other polynucleotides such as vectors, can be introduced into host cells, in culture or iin whole organisms. Introduced into host cells in culture or in whole organisms, such DNA, still would be isolated, as the term is used herein, because they would not be in their naturally occurring form or environment. Similarly, the polynucleotides may occur in a composition, such as a media formulations, solutions for introduction of polynucleotides, for examplE;, into cells, compositions or solutions for chemical or enzymatic reactions, for instance, which are not naturally occurring compositions, and, therein remain isolated polynucleotides within the meaning of that term as it is employed herein.
"Plasmids". Starting plasmids disclosed herein are either commercially available, publicly available, or can be constructed from available plasmids by routine application of well known, published procedures. Many plasmids and other cloning and expression vectors that can be used in accordance with the present invention are well known and readily available to those of skill in the art. Moreover, those of skill readily may construct any number of other plasmids suitable for use in the invention.
"Polynucleotides(s)" of the present invention may be in the form of RNA, such as mRNA, or in the form of DNA, including, for instance, cDNA and genomic DNA obtained by cloning or produced by chemical synthetic techniques or by a combination thereof. The DNA
may be double-stranded or single-stranded. Single-stranded polynucleotides may be the coding strand, also known as the sense strand, or it may be the non-coding strand, also referred to as the anti-sense strand. Polynucleotides generally refers to any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA.
Thus, for instance, polynucleotides as used herein refers to, among others, single-and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions or single-, double- and triple-stranded regions, single- and double-stranded RNA, and RNA
that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA
that may be single-stranded or, more typically, double-stranded, or triple-stranded, or a mixture of single- and double-stranded regions. In addition, polynucleotide as used herein refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA.
The strands in such regions may be from the same molecule or from different molecules. The regions may include all of one or more of the molecules, but more typically involve only a region of some of the molecules. One of the molecules of a triple-helical region often is an oligonucleotide. As used herein, the term polynucleotide also includes DNA or DNA that contain one or more modified bases. Thus, DNA or DNA with backbones modified for stability or for other reasons. are "polynucleotides" as that term is intended herein. Moreover, DNA or DNA comprising unusual bases, such as inosine, or modified bases, such as tritylated bases, to name just two examples, are polynucleotides as the term is used herein. It will be appreciated that a great variety of modifications have been made to DNA and RNA that serve many useful purposes known to those of skill in the art. The term polynucleotide as it is employed herein embraces such chemically, enzymatically or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including simple and complex cells, inter alia. Polynucleotides embraces short polynucleotides often referred to as oligonucleotide(s). It will also be appreciated that RNA
made by transcription of this. doubled stranded nucleotide sequence, and an antisense strand of a nucleic acid molecule of the invention or an oligonucleotide fragment of the nucleic acid molecule, are contemplated within the scope of the invention. An antisense sequence is constructed by inverting the sequence of a nucleic acid molecule of the invention, relative to its normal presentation for transcription. Preferably, an antisense sequence is constructed by inverting a region preceding the initiation codon or an unconserved region.
The antisense sequences may be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art.
"Stringent hybridization conditions" are those which are stringent enough to provide specificity, reduce the number of mismatches and yet are sufficiently flexible to allow formation of stable hybrids at an acceptable rate. Such conditions are known to those skilled in the art and are described, fo:r example, in Sambrook, et al, ( 1989). By way of example only, 1 S stringent hybridization with short nucleotides may be earned out at 5-10° below the TM using high concentrations of probe; such as 0.01-1.0 pmole/ml. Preferably, the term "stringent conditions" means hybridization will occur only if there is at least 95% and preferably at least 97% identity between the sequences.
"Variant(s)" of polynucleotides are polynucleotides that differ in nucleotide sequence from another, reference polynucle;otide. Generally, differences are limited so that the nucleotide sequences of the reference a.nd the variant are closely similar overall and, in many regions, identical. Changes in the nucleotide sequence of the variant may be silent.
That is, they may not alter the amino acids encoded by the polynucleotide. Where alterations are limited to silent changes of this type a variant will encode a polypeptide or polynucleotide with the same amino acid sequence as the reference. Changes in the nucleotide sequence of the variant may alter the amino acid sequence of a polypeptide encoded by the reference polynucleotide. Such nucleotide changes may result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptid.e or polynucleotide encoded by the reference sequence.
As hereinbefore mentioned, the present inventors have identified and sequenced a DNA
sequence encoding DHD. The DNA sequence is shown in the Sequence Listing as SEQ ID
NO:1 and N0:2.
It will be appreciated that the invention includes nucleotide or amino acid sequences which have substantial sequence homology with the nucleotide sequences shown in the Sequence Listing as SEQ ID NO:1 and N0:2. The term "sequences having substantial sequence homology" means those nucleotide and amino acid sequences which have slight or inconsequential sequence variations from the sequences disclosed in the Sequence Listing as SEQ ID NO:1 and N0:2 i.e. the homologous sequences function in substantially the same manner to produce substantially the same polypeptides as the actual sequences.
The variations may be attributable to local mutations or structural modifications. It is expected that a sequence having 85-90% sequence homology with the DNA sequence of the invention will provide a functional DHD polypeptide. Nucleic acid sequences having substantial sequence homology also include nucleic acid sequences having at least 70%, preferably at least 80%
homology with the nucleic acid sequence as shown in SEQ. ID. NO:1 and N0:2;
and fragments thereof having at least 15 to 50, preferably at least 15 bases, and preferably 20 to 30, which will hybridize to these sequences under stringent hybridization conditions.
The polynucleotides of the present invention may be employed as research reagents and materials for discovery of treatments of and diagnostics for disease, particularly human disease, as further discussed herein.
Analysis of the complete nucleotide and amino acid sequences of the protein of the invention using the procedures of Sambrook et al., supra, will be used to determine the expressed region, initiation codon and untranslated sequences of the DI-ID gene. The transcription regulatory sequences of the gene may be determined by analyzing fragments of the DNA for their ability to express a reporter gene such as the bacterial gene lacZ.
Primer extension using reverse transcriptase may also be used to determine the initiation site.
The nucleic acid molecules of the invention allow those skilled in the art to construct nucleotide probes for use in the detection of nucleotide sequences in biological materials. As shown in Figures 7 and 9 , a number of unique restriction sequences for restriction enzymes are incorporated in the nucleic acid molecule identified in the Sequence Listing as SEQ ID
NO:1 and NO: 2, and these provide access to nucleotide sequences which code for polypeptides unique to the DHI~ polypeptide of the invention. Nucleotide sequences unique to DHD or isoforms thereof=, can also be constructed by chemical synthesis and enzymatic ligation reactions earned out: by procedures known in the art.

A nucleotide probe may be Labeled with a detectable marker such as a radioactive label which provides for an adequate signal and has sufficient half life such as 32p, 3H, 14C or the like.
Other detectable markers which may be used include antigens that are recognized by a specific labeled antibody, fluorescent: compounds, enzymes, antibodies specific for a labeled antigen, and chemiluminescent compounds. An appropriate label may be selected having regard to the rate of hybridization and binding of the probe to the nucleotide to be detected and the amount of nucleotide available for hybridization. The nucleotide probes may be used to detect genes related to or analogous to DHD of the invention.
Accordingly, the present invention also provides a method of detecting the presence of nucleic acid molecules encoding a p~olypeptide related to or analogous to DHD in a sample comprising contacting the sample under hybridization conditions with one or more of the nucleotide probes of the invention labeled with a detectable marker, and determining the degree of hybridization between the nucleic acid molecule in the sample and the nucleotide probes.
Hybridization conditions which may be used in the method of the invention are known in the art and are described for example in Sambrook J, et al., supra. The hybridization product may be assayed using techniques known in the art. The nucleotide probe may be labeled with a detectable marker as described herein and the hybridization product may be assayed by detecting the detectable marker or the detectable change produced by the detectable marker.
The nucleic acid molecule of the invention also permits the identification and isolation, or synthesis of nucleotide sequences which may be used as primers to amplify a polynucleotide molecule of the invention, for example in polymerase chain reaction (PCR). The length and bases of the primers for use in the PCR are selected so that they will hybridize to different strands of the desired sequence and at relative positions along the sequence such that an extension product synthesized from one primer when it is separated from its template can serve as a template for extension of the other primer into a nucleic acid of defined length.
S
Primers which may be used in the invention are oligonucleotides i.e. molecules containing two or more deoxyribonucleotides of the nucleic acid molecule of the invention which occur naturally as in a purified restriction endonuclease digest or are produced synthetically using techniques known in the art such as, for example, phosphotriester and phosphodiester methods (See Good et al, 1977) or automated techniques (see, for example, Conolly, B.
A., 1987).
The primers are capable of acting as a point of initiation of synthesis when placed under conditions which permit the synthesis of a primer extension product which is complementary to the DNA sequence of the invention e.g. in the presence of nucleotide substrates, an agent for polymerization such as DNA polymerase and at suitable temperature and pH.
Preferably, the primers are sequences that do not form secondary structures by base pairing with other copies of the primer or sequences that form a hair pin configuration. The primer may be single or double-stranded. When the primer is double-stranded it may be treated to separate its strands before using it to prepare amplification products. The primer preferably contains between about 7 and 25 nucleotides.
The primers may be labeled with detectable markers which allow for detection of the amplified products. Suitable detectable markers are radioactive markers such as P-32, S-35, I-125, and H-3, luminescent markers such as chemiluminescent markers, preferably luminol, and Iluorescent markers, preferably dansyl chloride, fluorcein-5-isothiocyanate, and 4-fluor-7-nitrobenz-2-axa-1,3 diazole, enzyme markers such as horseradish peroxidase, alkaline phosphatase, .beta.-galactosidase, acetylcholinesterase, or biotin.
It will be appreciated that the primers may contain non-complementary sequences provided that a sufficient amount of the primer contains a sequence which is complementary to a nucleic acid molecule of the invention or oligonucleotide sequence thereof, which is to be amplified.
Restriction site linkers may also be incorporated into the primers allowing for digestion of the amplified products with the appropriate restriction enzymes facilitating cloning and sequencing of the amplified product.
Thus, a method of determining the presence of a nucleic acid molecule having a sequence encoding DHD or a predetermined oligonucleotide fragment thereof in a sample, is provided comprising treating the sample with primers which are capable of amplifying the nucleic acid molecule or the predetermined oligonucleotide fragment thereof in a polymerase chain reaction to form amplified sequences., under conditions which permit the formation of amplified sequences and, assaying for amplified sequences.
The polymerase chain reaction refers to a process for amplifying a target nucleic acid sequence as generally described in Innis et al, Academic Press, 1989, in Mullis el al., U.S. Pat. No.
4,863,195 and Mullis, U.S. Pat. No. 4,683,202 which are incorporated herein by reference.
Conditions for amplifying a nucleic acid template are described in M. A. Innis and D. H.
Gelfand, 1989, which is also incorporated herein by reference.

The amplified products can be isolated and distinguished based on their respective sizes using techniques known in the art. For example, after amplification, the DNA sample can be separated on an agarose gel and visualized, after staining with ethidium bromide, under ultra violet (UV) light. DNA may be amplified to a desired level and a further extension reaction may be performed to incorporate nucleotide derivatives having detectable markers such as radioactive labeled or biotin labeled nucleoside triphosphates. The primers may also be labeled with detectable markers. The detectable markers may be analyzed by restriction and electrophoretic separation or other techniques known in the art.
The conditions which may bE; employed in the methods of the invention using PCR are those which permit hybridization and amplification reactions to proceed in the presence of DNA in a sample and appropriate complementary hybridization primers. Conditions suitable for the polymerise chain reaction are generally known in the art. For example, see M.
A. Innis and D.
H. Gelfand, 1989, which is incorporated herein by reference. Preferably, the PCR utilizes polymerise obtained from the thermophilic bacterium Thermus aquatics (Taq polymerise, GeneAmp Kit, Perkin Elmer Cetus) or other thermostable polymerise may be used to amplify DNA template strands.
It will be appreciated that other techniques such as the Ligase Chain Reaction (LCR) and Nucleic-Acid Sequence Based Amplification (NASBA) may be used to amplify a nucleic acid molecule of the invention. In LCR, two primers which hybridize adjacent to each other on the target strand are ligated in the presence of the target strand to produce a complementary strand (Barney, 1991 and European Published Application No. 0320308, published Jun. 14, 1989). NASBA is a continuous amplification method using two primers, one incorporating a promoter sequence recognized by an RNA polymerase and the second derived from the complementary sequence of the target sequence to the first primer (U.S. Ser.
No. 5,130,238 to Malek).
The present invention also teaches vectors which comprise a polynucleotide or polynucleotides of the present invention, host cells which are genetically engineered with vectors of the invention and the production of polynucleotides of the invention by recombinant techniques.
In accordance with this aspect of the invention the vector may be, for example, a plasmid vector, a single or double-stranded phage vector, a single or double-stranded RNA or DNA
viral vector. In certain preferred embodiments in this regard, the vectors provide for specific expression. Such specific expression may be inducible expression or expression only in certain types of cells or both inducible and cell-specific. Particularly preferred among inducible vectors are vectors that can be induced for expression by environmental factors that are easy to manipulate, such as temperature and nutrient additives. A variety of vectors suitable to this aspect of the invention, including constitutive and inducible expression vectors for use in prokaryotic and eukaryotic hosts, are well known and employed routinely by those of skill in the art. Such vectors includE:, among others, chromosomal, episomal and virus-derived vectors, e.g., vectors derived from bacterial plasmids, from bacteriophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as S V40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids, all may be used for expression in accordance with this aspect of the present invention.
The following vectors, which are commercially available, are provided by way of example.
Among vectors preferred for use in bacteria are pQE70, pQE60 and pQE-9, available from Qiagen; pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNHl6a, pNHl8A, pNH46A, available from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRITS
available from Pharmacia, and pBR322 (ATCC 37017). Among preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXTI and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia. These vectors are listed solely by way of illustration of the many commercially available and well known vectors that are available to those of skill in the art for us,e in accordance with this aspect of the present invention. It will be appreciated that any other plasmid or vector suitable for, for example, introduction, maintenance, propagation or expression of a polynucleotide or polypeptide of the invention in a host may be used in this aspect of the invention. Generally, any vector suitable to maintain, propagate or express polynucleotides to express a polypeptide or polynucleotide in a host may be used for expression in this regard.
The appropriate DNA sequence may be inserted into the vector by any of a variety of well-known and routine techniques. In general, expression constructs will contain sites for transcription initiation and termination, and, in the transcribed region, a ribosome binding site for translation. The coding portion of the mature transcripts expressed by the constructs will include a translation initiating AUG at the beginning and a termination codon appropriately positioned at the end of the polynucleotide to be translated.
The DNA sequence in the e~;pression vector is operatively linked to appropriate expression control sequence(s), including, for instance, a promoter to direct mRNA
transcription.
Promoter regions can be selected from any desired gene using vectors that contain a reporter transcription unit lacking a promoter region, such as a chloramphenicol acetyl transferase ("CAT") transcription unit, downstream of restriction site or sites for introducing a candidate promoter fragment; i.e., a fragment that may contain a promoter. As is well known, introduction into the vector of a promoter-containing fragment at the restriction site upstream of the cat gene engenders production of CAT activity, which can be detected by standard CAT
assays. Vectors suitable to this end are well known and readily available, such as pKK232-8 and pCM7. Promoters for expression of polynucleotides of the present invention include not only well known and readily available promoters, but also promoters that readily may be obtained by the foregoing technique, using a reporter gene. Amang known prokaryotic promoters suitable for expression of polynucleotides and polypeptides in accordance with the present invention are the E. coli lacI and lacZ and promoters, the T3 and T7 promoters, the gpt promoter, the lambda Ply, PL promoters and the trp promoter. Among known eukaryotic promoters suitable in this regard are the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, the promoters of retroviral LTRs, such as those of the Rous sarcoma virus ("RSV"), and metallothionein promoters, such as the mouse metallothionein-I promoter.
Vectors for propagation and expression generally will include selectable markers and amplification regions, such as, for example, those set forth in Sambrook et al., supra.

As hereinbefore mentioned, 'the present invention also teaches host cells which are genetically engineered with vectors of the invention.
Polynucleotide constmcts in host cells can be used in a conventional manner to produce the gene product encoder by the recombinant sequence. The DHD polynucleotide or polypeptide products or isoforms or parts thereof, may be obtained by expression in a suitable host cell using techniques known in the art. Suitable host cells include prokaryotic or eukaryotic organisms or cell lines, for example bacterial, mammalian, yeast, or other fungi, viral, plant or insect cells. Method;. for transforming or transfecting cells to express foreign DNA are well known in the art (See: for example, Itakura et al., U.S. Pat. No. 4,704,362;
Hinnen et al., 1978;
Murray et al., U.S. Pat. No. 4,801,542; Upshall et al., U.S. Pat. No.
4,935,349; Hagen et al., U.S. Pat. No. 4,784,~~50; A~;el et al., U.S. Pat. No. 4,399,216; Goeddal et al., U.S. Pat. No.
4,766,075; and Sambrook et al, 1989, all of which are incorporated herein by reference).
1 S Representative examples of appropriate hosts include bacterial cells, such as streptococci, staphylococci, E. coli, streptomyces and Bacillus subtilis cells; fungal cells, such as yeast cells and Aspergillus cells: insect cells such as Drosophila S2 and Spodoptera S~
cells; animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, 293 and Bowes melanoma cells; and plant cells.
Host cells can be genetically engineered to incorporate polynucleotides and express polynucleotides of the present invention. Introduction of a polynucleotides into the host cell can be affected by calcium phosphate transfection, DEAF-dextran mediated transfection, transvection, microinjection, cationic lipid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction, infection or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al. (1986) and Sambrook et al. ( 1989).
As hereinbefore mentioned, the present invention also teaches the production of polynucleotides of the invention by recombinant techniques.
The DHD polynucleotides may encode a polypeptide which is the mature protein plus additional amino or carboxyl-terminal amino acids, or amino acids interior to the mature a polypeptide (when the mature form has more than one polypeptide chain, for instance). Such sequences may play a role in processing of a protein from precursor to a mature form, may allow protein transport, may lengthen or shorten protein half life or may facilitate manipulation of a protein for assay or production, among other things. As generally is the case in vivo, the additional amino acids may be processed away from the mature protein by cellular enzymes.
A precursor protein, having 'the mature form of the polypeptide fused to one or more prosequences may be an inactive form of the polypeptide. When prosequences are removed such inactive precursors generally are activated. Some or all of the prosequences may be removed before activation. Generally, such precursors are called proproteins.
In sum, a polynucleotide of the present invention may encode a mature protein, a mature protein plus a leader sequence (which may be referred to as a preprotein), a precursor of a mature protein having one or more prosequences which are not the leader sequences of a preprotein, or a preproprotein, which is a precursor to a proprotein, having a leader sequence and one or more prosequences, which generally are removed during processing steps that produce active and mature forms of the polypeptide.
The polypeptides of the invention may be prepared by culturing the host/vector systems described above, in order to express the recombinant polypeptides.
Recombinantly produced DHD based protein or parts thereof, may be further purified using techniques known in the art such as commercially available protein concentration systems, by salting out the protein followed by dialysis, by ai~mity chromatography, or using anion or cation exchange resins.
Mature proteins can be expressed in mammalian cells, yeast, bacteria, or other cells under the control of appropriate promoters. Cell-free translation systems can also be employed to produce such proteins using DNA derived from the DNA constructs of the present invention.
Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described by Sambrook et al., supra.
Polynucleotides of the invention, encoding the heterologous structural sequence of a polynucleotide or polypeptide of the invention generally will be inserted into a vector using standard techniques so that it is operably linked to the promoter for expression. The polynucleotide will be positioned so that the transcription start site is located appropriately 5' to a ribosome binding site. 7.'he ribosome binding site will be 5' to the AUG
that initiates translation of the polynucleotide or polypeptide to be expressed. Generally, there will be no other open reading frames that begin with an initiation codon, usually AUG, and lie between the ribosome binding site and the initiation codon. Also, generally, there will be a translation stop codon at the end of the expressed polynucleotide and there will be a polyadenylation signal in constructs for use in eukaryotic hosts. Transcription termination signal appropriately disposed at the 3' end of the transcribed region may also be included in the polynucleotide construct.
S
For secretion of the translated protein into the lumen of the endoplasmic reticulum, into the pen-iplasmic space or into the extracellular environment, appropriate secretion signals may be incorporated into the expressed polynucleotide or polypeptide. These signals may be endogenous to the polynucleotide or they may be heterologous signals.
Microbial cells employed in expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents, such methods are well know to those skilled in the art. DHD polynucleotide or polypeptide can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography is employed for purification. Well known techniques for refolding protein may be employed to regenerate active conformation when the polynucleotide is denatured during isolation and or purification.
In a preferred embodiment, a nucleic acid molecule of the invention may be cloned into a glutathione S-transferase (GST) gene fusion system for example the pGEX-1 T, pGEX-2T
and pGEX-3X of Pharmacia. The fizsed gene may contain a strong lac promoter, inducible to a high level of expression by IPTG, as a regulatory element. Thrombin or factor Xa cleavage sites may be present which allow proteolytic cleavage of the desired polypeptide from the fusion product. The glutathiane S-transferase-DHD fusion protein may be easily purified using a glutathione sepharose 4B column, for example from Pharmacia. The 26 kd glutathione S-transferase polypeptide can be cleaved by thrombin (pGEX-1 or pGEX-2T) or factor Xa (pGEX-3X) and resolved from the using the polypeptide using the same affinity column.
Additional chromatographic steps can be included if necessary, for example Sephadex or DEAE cellulose. The two enzymes may be monitored by protein and enzymatic assays and purity may be confirmed using SDS-PAGE.
The DHD protein or parts thereof may also be prepared by chemical synthesis using techniques well known in the: chemistry of proteins such as solid phase synthesis (Mernfield, 1964) or synthesis in homogenous solution (Houbenweyl, 1987).
Within the context of the present invention, DHD polypeptide includes various structural forms of the primary protein which retain biological activity. For example, DHD polypeptide may be in the form of acidic or basic salts or in neutral form. In addition, individual amino acid residues may be modified by oxidation or reduction. Furthermore, various substitutions, deletions or additions may bf; made to the amino acid or nucleic acid sequences, the net effect being that biological activity of DHD is retained. Due to code degeneracy, for example, there may be considerable variation in nucleotide sequences encoding the same amino acid.
The polypeptide may be expressed in a modified form, such as a fusion protein, and may include not only secretion signals but also additional heterologous functional regions. Thus, for instance, a region of additional amino acids, particularly charged amino acids, may be added to the C- or N-terminus of the polypeptide to improve stability and persistence in the host cell, during purification or during subsequent handling and storage.
Also, fusion proteins may be added to the polynucleotide or polypeptide to facilitate purification.
Such regions may be removed prior to final preparation of the polynucleotide or polypeptide.
The addition of peptide moieties to polynucleotide or polypeptides to engender secretion or excretion, to improve stability or to facilitate purification, among others, are familiar and routine techniques in the art. In drug discovery, for example, proteins have been fused with antibody Fc portions for the purpose of high-throughput screening assays to identify antagonists (see Bennett et al., 1995, and Johanson et al.,1995).
This invention is also related to the use of the DHD polynucleotides to detect complementary polynucleotides as a diagnostic reagent.
1 S Detection of the level of expression of DHD in a eukaryote, particularly a mammal, and especially a human, will provide a diagnostic method for diagnosis of a disease. Eukaryotes (herein also "individual(s)"), particularly mammals, and especially humans, exhibiting decreased levels of DHD may be detected by a variety of techniques. Nucleic acids for diagnosis may be obtained from an infected individual's cells and tissues, such as the striatum, nucleus accumbens and olfactory tubercule. RNA may be used directly for detection or may be amplified enzymatically by using PCR (Saiki et al., 1986) prior to analysis. As an example, PCR primers complementan~ to the nucleic acid encoding DHD can be used to identify and analyze DHD presence and/or expression. Using PCR, characterization of the level of DHD
present in the individual may be made by comparative analysis.

The invention thus provides a process for diagnosing disease by using methods known in the art and methods described herein to detect decreased expression of DHD
polynucleotide. For example, decreased expression of DHD polynucleotide can be measured using any on of the methods well known in the art for the quantification of polynucleotides, such as, for example, PCR, RT-PCR, DNAse protection, northern blotting and other hybridization methods. Thus, the present invention provides a method for diagnosing triplet-repeat disorders, and a method for diagnosing a genetic pre--disposition for triplet-repeat disorders and other disorders of the basal ganglia including schizophrenia, stroke, trauma, Parkinson's disease and Alzheimer's disease (AD). More generally, the present invention provides a method for diagnosing a genetic pre-disposition for neurological disorders characterized by progressive cell loss.
The invention also provides a method of screening compounds to identify those which enhance (agonist) or block (antagonist) the action of DHD polypeptides or polynucleotides, such as its interaction with DHD-binding molecules. The identification of mutations in specific genes in inherited neurodegenerative disorders, combined with advances in the field of transgenic methods, provides those of skill in the art with the information necessary to further study human diseases. This is extraordinarily usefizl in modeling familial forms of triplet-repeat disorders and other disorders of the basal ganglia including schizophrenia, stroke, trauma, Parkinson's disease and Alzheimer's disease (AD). More generally, the present invention is usefirl for modeling neurological disorders characterized by progressive cell loss, as well as those involving acute cell loss, such as stroke and trauma.

For example, to screen for agonists or antagonists, a synthetic reaction mix, a cellular compartment, such as a membrane, cell envelope or cell wall, or a preparation of any thereof, may be prepared from a cell that expresses a molecule that binds DHI~. The preparation is incubated with labeled DHD in the absence or the presence of a candidate molecule which may be a DHI~ agonist or antagonist. The ability of the candidate molecule to bind the binding molecule is reflected in decreased binding of the labeled ligand.
DHD-like erects of potential agonists and antagonists may by measured, for instance, by determining activity of a reporter system following interaction of the candidate molecule with a cell or appropriate cell preparation, and comparing the effect with that of DHD or molecules that elicit the same effects as DHD. Reporter systems that may be useful in this regard include, but are not limited to, colorimetric labeled substrate converted into product, a reporter gene that is responsive to changes in DHD activity, and binding assays known in the art.
Another example of an assay for DHD antagonists is a competitive assay that combines DHD
and a potential antagonist with membrane-bound DID-binding molecules, recombinant DHI~
binding molecules, natural substrates or ligands, or substrate or ligand mimetics, under appropriate conditions for a competitive inhibition assay. DI-ID can be labeled, such as by radioactivity or a colorimetri.c compound, such that the number of DHD
molecules bound to a binding molecule or converted to product can be determined accurately to assess the effectiveness of the potential antagonist.
Potential antagonists include small organic molecules, peptides, polypeptides and antibodies that bind to a polynucleotide or polypeptide of the invention and thereby inhibit or extinguish its activity. Potential antagonists also may be small organic molecules, a peptide, a polypeptide such as a closely related protein or antibody that binds the same sites on a binding molecule, such as a binding molecule, 'Nithout inducing DHD-induced activities, thereby preventing the action of DI-~ by excluding DID from binding.
Potential antagonists include a small molecule which binds to and occupies the binding site of the polypeptide thereby preventing binding to cellular binding molecules, such that normal biological activity is prevented. Examples of small molecules include but are not limited to small organic molecules, peptides or peptide-like molecules. Other potential antagonists include antisense molecules see Okano, 1988, for a description of these molecules). Preferred potential antagonists include compounds related to and derivatives of DHD.
Thus, the present invention provides a method for screening and selecting compounds which promote triplet-repeat disorders, and a method for screening and selecting compounds which treat or inhibit triplet-repeat disorders, as well as schizophrenia, stroke, trauma, Parkinson's disease and Alzheimer's disease. More generally, the present invention provides a method for screening and selecting compounds which promote or inhibit neurological disorders characterized by progressive; cell loss, as well as those involving acute cell loss, such as stroke and trauma.
The selected antagonists and agonists may be administered, for instance, to inhibit progressive and acute neurological disorders, such as Huntington's disease, schizophrenia, Alzheimer's disease (AD), stroke or trauma.
Antagonists and agonists and other compounds of the present invention may be employed alone or in conjunction with other compounds, such as therapeutic compounds.
The pharmaceutical compositions. may be administered in any effective, convenient manner including, for instance, administration by direct microinjection into the affected area, or by intravenous or other routes. These compositions of the present invention may be employed in combination with a non-sterile or sterile carrier or carriers for use with cells, tissues or organisms, such as a pharmaceutical carrier suitable for administration to a subject. Such compositions comprise, for instance, a media additive or a therapeutically effective amount of antagonists or agonists of the invention and a pharmaceutically acceptable carrier or excipient.
Such carriers may include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol and combinations thereof. The formulation is prepared to suit the mode of administration.
The invention further provides diagnostic and pharmaceutical packs and kits comprising one or more containers filled with one or more of the ingredients of the aforementioned compositions of the invention. Associated with such containers) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, reflecting approval by the agency of the manufacture, use or sale of the product for human administration.
The pharmaceutical compositions generally are administered in an amount effective for treatment or prophylaxis of a. specific indication or indications. It is appreciated that optimum dosage will be determined by standard methods for each treatment modality and indication, taking into account the indication, its severity, route of administration, complicating conditions and the like. In therapy or a;; a prophylactic, the active agent may be administered to an individual as an injectable composition, for example as a sterile aqueous dispersion, preferably isotonic. For administration to mammals, and particularly humans, it is expected that the daily dosage level of the active agent will be from 0.001 mg/kg to 10 mg/kg, typically around 0.01 mg/kg. The physician in any event will determine the actual dosage which will be most suitable for an individual and will vary with the age, weight and response of the particular individual.
The above dosages are exemplary of the average case. There can, of course, be individual instances where higher or lower dosage ranges are merited, and such are within the scope of this invention.
EXAMPLES
The present invention is further described by the following examples. These examples, while illustrating certain specific aspects of the invention, do not portray the limitations or circumscribe the scope of the disclosed invention.
EXAMPLE 1 - Isolation of DHD
Wild-type (B6CBAF1) and l-ID transgenic [B6CBA-TgN(Hdexonl)62Gpb] mice (Jackson Laboratories) and adult Sprague-Dawley rats (250-300 g; Charles River Laboratories) and were used in this study. The genotype of the mice was determined by PCR
amplification of a 100 by region of the integrated human HD exon 1 transgene using primers corresponding to nts 3340-3459 (5'-AGG GCT GTC AAT CAT GCT GG-3') and nts 3836-3855 (5'-AAA
CTC ACG GTC GGT GCA GC-3') of clone E4.1 of the human HD gene (Accession number L34020). PCR conditions used are described in Mangiarini et al.(1996). DNA was extracted from a tail clip and an ear punch from each mouse used in this study. Both samples were subjected to PCR genotype analysis. For in situ hybridization analysis, the animals were anesthetized with >100 mg/kg sodium pentobarbital, decapitated, the brains removed and stored at -70°C prior to sectioning. For RNA isolation, animals were anesthetized, decapitated and the striatum and cortex were excised and stored in liquid nitrogen prior to RNA extraction. Animal care was given according to protocols approved by Dalhousie University and the Canadian Council of Animal Care.
Differential display was used to identify novel mDNA or previously described mDNA whose relative expression levels are altered as a result of the presence of the transgene. Using differential display, the mRNA populations derived from the striatum of 10 week old wild type were compared with age-matched R6/2 transgenic mice. Differential display has been used extensively (> 750 references) since its development (Lung and Pardee, 1992) to identify changes in gene expression in cells and in tissues including brain (Douglass et al., 1995; Babity et al., 1997a; Livesey et al., 1997; Berke et al., 1998). Perhaps the most important finding was the demonstration by Qu et al., ( 1996) that differential display can be used to isolate genes differentially expressed in inbred strains of mice. The power of differential display is that the sequence information obtained can be directly related to the experimental paradigm.
Moreover, such sequence information includes sufficient information to identify transcripts and can then lead to experiments that reveal function of the cognate protein in the experimental model.
DNA sequence information of potentially differentially expressed cDNA can be used to S generate oligonucleotide probes for in situ hybridization to define the anatomical and temporal patterns of expression of specific transcripts (see Babity et al., 1997a).
This technique is especially useful to study ch<~nges in steady-state levels of mRNA in heterogeneous tissue such as brain. Brain tissue can be micro-dissected (Babity et al., 1997b). This enabled the present inventors to reduce the requirement for tissue, and hence compare the mRNA
populations derived from individual animals for each experimental group Thus RT-PCR (Denovan-Wright et al., 1999) was used to identify differences in the patterns of gene expression between the striatum of wild-type and transgenic mice that were hemizygous for the 5' UTR, exon 1 and part of intron 1 of the human Huntingon's Disease gene. Total cellular RNA was isolated from the striatum and cortex of three 10 week-old wild-type and three 10 week-old R6/2 HD mice (Mangiarini et al., 1996) and used as the template to generate single-stranded cDNA. Total cellular RNA from each animal and tissue was purified using TrizolT"" reagent (Gibco BRL) and the manufacture's protocol. 10 pg aliquots of total RNA were 'treated with RQ 1 DNAse-free DNAse (Promega) in the presence of DNAsinT"' (Promega) DNAse inhibitor to remove trace genomic DNA and then converted to single-stranded cDNA. The primers and conditions for PCR amplification follow those of the Deltas RNA fingerprynting manual (Clontech).

The eDNA was then used as the substrate for PCR reactions using 57 differential display primer combinations. The radio-labelled PCR products were fractionated on a denaturing acrylamide sequencing gels using a Genomyx LRT"" sequencing apparatus, transferred to 3MM
filter paper and dried. The dried acrylamide gels were exposed to autoradiography film (BioMax MRS) overnight. .After fractionating the radio-labelled PCR products on denaturing acrylamide gels, it was found that the overwhelming majority of the approximately 18,000 PCR products screened were common to both the wild-type and HD mice (data not shown).
One PCR product, amplified using the primers P7 (5'-ATT AAC CCT CAC TAA ATG
CTG
TAT G- 3') and T6 (5'- CA'T TAT GCT GAG TGA TAT CTT TTT TTT TCG- 3') of approximately 500 bp, was observed in each of three samples derived from the striatum of wild-type mice (Fig. lA). This S00 by band was absent from the samples derived from the striatum of the HD mice (Fig. 1 A) and was absent from each of the samples derived from the cortical tissue (data not show).
Figure lA shows the Down-regulated in Huntington's Disease (DHD) transcript, identified by differential display RT PCR. A band of approximately 500 by (arrow) was amplified from cDNA made form 10 week-old wild-type but not 10 week-old HD striatal tissue.
Total RNA
from individual animals (numbered 1-6) was used as the substrate for the generation of single-stranded cDNA. Animals 1, 2 and 3 were transgenic HD mice. Animals 4, 5 and 6 were wild-type mice.

EXAMPLE 2 - Cloning of I)HD
The 500 by band, designate DHDpcr, was excised from the dried gel and rehydrated in 40 pl of H20 for 10 min at room temperature. The eluted DNA was subjected to PCR re-amplification using the P7 and T6 primers, rTaq polymerase (Pharmacia) and the following conditions: 60" @ 94°C, 19 :~c (30" @ 94°C, 30" @ 58°C, 120" @ 68°C + 4" per cycle), 7' @
68°C. The PCR reaction was subjected to agarose gel electrophoresis and the 500 by band was removed from the gel, extracted from the agarose using the Qiagen gel extraction protocol and cloned into the vector, pGem-T using standard methods. Plasmid DNA was isolated from selected transfi~rmants using Qiagen spin columns. 'The resultant clone was named pDHD.
EXAMPLE 3 - Identification of DHD
The cloned insert of pDHD was radio-labelled and used as a hybridization probe in northern blot analysis (Fig. 1B). Northern blots of total RNA were prepared using the method described in Denovan-Wrigl~t et al. (1998). The 500 by cloned insert of DHD
was radio-labelled with [a-32P]dCTP (3000 Ci/mmol) using the Ready-to-Go dCTP beads (Pharmacia).
Northern blot hybridization, brain tissue preparation and irr situ hybridization are described in Denovan-Wright et al. (1998). The S00 by cloned insert of pDHD annealed to a transcript of approximately 7.5 kb in total RNA isolated from the striatum of ten week-old wild-type mice.

Figure 1B demonstrates that DHD is expressed in the striatum but not the cortex of wild-type mice and the steady-state levels of DHD are reduced in 10 week ald transgenic HD mice. The differential expression of DHD in HD mice was confirmed by northern blot analysis. The cloned insert of pDHD was radio-labelled and used as a hybridization probe in northern blot analysis. The northern blot was prepared by size-fractionating total RNA from the striatum and cortex of three individual 10 week-old HD ( 1, 2 and 3) and wild-type (4, 5 and 6) mice.
Following the hybridization of pDHD, the radio-label was removed and the blot was subsequently allowed to hybridize with a probe that detects constituitively expressed cyclophilin. The hybridization pattern of the cyclophilin probe is aligned below the northern blot demonstrating that equivalent amount of RNA were present in each lane.
The relative mobility of RNA molecular weight standards (RNA ladder, Gibco BRL) are shown on the left of the northern blot.
The hybridization signal of pDHD was significantly lower in the RNA samples derived from the striatum of 10 week-old :EiD mice. No expression of the DHD mRNA was detected in the cortical RNA samples derived from either the wild-type or HD mice.
EXAMPLE 4 - Sequencing DHD
The sequence of the cloned dii~erential display band, pDHD, was determined using M13 universal forward and reverse sequencing primers and the T7 sequencing kit (Pharmacia). The 484 by cDNA fragment did not have sequence similarity to any Genbank entries.

Figure 2 shows the nucleotide sequence of the cloned DHD differential display product, pDHD. The position of the primers used to amplify the fragment are underlined and labelled.
The nucleotide sequence and position of oligonucleotide probes 1 and 2 within the pDHI~
sequence are shown.
EXAMPLE 5 - Localization of DHD
In order to identify the coding strand and to localize the transcript in the wild-type mouse brain, two oligonucleotide probes were designed (probe 1, 5'- GAA CAT GTA GCA
TAT
ACT CCA GAC AAC AGA TCA TAT GG - 3'; probe 2, 5' - CAG CTT CTC CAC AGG
AAC ACA GTA ACA AAG AG -3') that were complementary to different regions and strands of the 484 by pDHD clone. These oligonucleotides were used for in situ hybridization analysis. Using high stringency post in situ hybridization washes (2 x 30' in 1X SSC @ 58oC, 4 x 15' in 1X SSC @ 58oC, 4 x 15' in O.SX SSC @ 58oC, 4 x 15' in 0.25X SSC @
58oC), it was found that oligonucleotide probe 1 annealed with mltNA in the striatum, nucleus accumbens and olfactory tubercule of ten week-old wild-type mice (Fig. 3A).
The hybridization signal was significantly reduced in the striatum, nucleus accumbens and olfactory tubercle of the 10 week-old I-ID mice (Fig. 3A).
Figure 3A shows in situ hybridization of probe 1 to coronal (top three sections) and saggital (bottom section) 10 week-old wild-type (WT) and HD mouse brain sections.
Specific hybridization of the probe was observed in the striatum, nucleus accumbens and olfactory tubercle of wild-type mice. 'The top three sections represent the distribution of DHD
throughout the rostral-caudal axis of the striatum.
The in situ hybridization results confirmed the northern blot analysis demonstrating, 1 ) that the expression of DHD mRNA was restricted to the striatum, nucleus accumbens and olfactory tubercle and 2) that the levels of DHI~ mRNA were decreased in I~ mice compared to the wild-type. The probe did nol: anneal with mRNA in any other brain nuclei. No hybridization of oligonucleotide probe 2 was observed in any region of the brain in wild-type or HD mice (Fig.
3). Based on this hybridizatiion, the coding strand, complementary to probe l, of pDHD was defined.
EXAMPLE 6 - Characterization of DHD
The in situ hybridization using oligonucleotide probe 1 demonstrated that DHD
mRNA levels 1 S in the striatum , nucleus accumbens and olfactory tubercule were decreased in ten week- old HD mice. By ten weeks of age, the HD mice all showed motor symptoms including resting tremor and stereotypic involuntary movements. Moreover, these mice immediately clasped their feet together and curled into a tight ball when picked up by their tails.
As the phenotypic signs are progressive over a number of weeks, the present inventors examined whether the DHD transcript was ever expressed in the striatum of the HD mice or whether the steady-state levels of the transcript diminished in the striatum in a course that parallelled the development of the motor disorders. Wild-type and HD mice were sacrificed at 5, 7 and 8 weeks of age and their brains were prepared for in situ hybridization analysis using probe 1 (Fig. 3B).
Figure 3B shows the levels of DHD mRNA decrease in HD mice over the period of time that the HD mice develop abnormal movements and postures. In situ hybridization analysis of coronal and saggital sections of wild-type and HD mouse brain using oligonucleotide probe 1 which is complementary to the coding strand of DHD. At 5 weeks of age, before the development of motor symptoms, the HD mice express the DHD transcript in the same brain nuclei and at the same relative levels as wild-type mice. The steady-state level DHD decreases in the striatum, nucleus accumbens and olfactory tubercle from 5 to 10 weeks in the HD but not wild-type mice. By 9 wf;eks of age, the HD mice have abnormal movement and posture.
The numbers refer to the age; in weeks of the wild-type (WT) and Huntington's (HD) transgemc mice.
None of the mice at these ages had overt motor symptoms. Sections taken throughout the rostral-caudal axis of the striatum showed that DHD was expressed in the 5 week-old wild-type and HD mice. The relative hybridization of probe 1 did not change in 5, 7, 8 and 10 week-old wild-type mice. The intensity of the hybridization signal appeared to decrease in the striatum, nucleus accumbens and olfactory tubercle of HD mice from S to 10 weeks compared to their wild-type litter mates (Fig. 3B).
One day old wild-type and HD mice were frozen, sectioned on a cryostat and whole mouse sections were prepared for i~r situ hybridization using probe 1. The same high stringency post-hybridization washing conditions were employed for the one day-old mouse body sections as were used for the adult mouse brain sections. Parallel in situ hyridization experiments using the probe :2 were performed in order to determine the level of non-specific signal in the mouse sections. Probe 1 specifically annealed to the developing striatum (Fig.
3C).
Figure 3C demonstrates that DHD is expressed in the developing striatum of one day-old wild-type and HD mice. The: sections on the left were subjected to in situ hybridization using probe 1. Following hybridization, the sections were counter-stained with cresyl violet to visualize the mouse organs. The signal outside the brain was non-specific as probe 2 and other unrelated control oligonucleotide probes all labelled these tissues.
There was no difference in the pattern of hybridization between the one day-old wild-type and HD mice demonstrating that DHD was expressed in the developing brain of both wild-type 1 S and HD mice.
Following in situ hybridization, the sections were covered in autoradiographic emulsion, left in the dark to expose for 4 weeks and then developed and viewed under dark-field microscopy or, after counter-staining the sections with cresyl violet to visualize neuronal cell bodies, under bright-field microscopy. Silver grains were observed to be concentrated in the striatum of the wild-type mice. Figure 3D shows emulsion autoradiography of mouse brain sections following in situ hybridization of probE; 1 demonstrated that the DHD transcript is expressed in neurons.
DHD is not homogeneously distributed throughout the mouse striatum. Dark field illumination of the sections after emulsion autoradiography showed that the silver grains were clustered in specific regions of the 10 week old wild-type mouse striatum (A
and C). Sections from 10 week old HD mice subjected to identical in situ and emulsion autoradiographic conditions are shown in B and D. The photomicrographs shown in A and B were viewed using the lOX objective (bar represents 100 Vim). The micrographs shown in C
and D, were viewed under the 20X objective (bar represents 25 Vim). The insert in panel C
is a portion of the section in A and C counter-stained with cresyl violet to visualize the neurons, viewed using the 40X objective under bright filed illumination. Note the distribution of the silver grains over some, but not all, of the: striatal neurons as well as being concentrated around clusters of neurons. It appeared that the silver grains were absent from fibre tracks within the striatum.
It appeared that DHD mRNA was not confined to regions close to the nucleus but was dispersed in cellular processes.
EXAMPLE 7 - Confirmation of DHD in other Mammalian Species (Rat) The oligonucleotide (probe 1. ) complementary to the coding strand of the DHD
transcript, was also used as an in sitzs hybridization probe against coronal brain sections derived from adult rats. Figure 4 shows in situ hybridization analysis of adult rat brain sections using oligonucleotide probe 1 complementary to the coding-strand of DHI~ revealed that the pattern of expression of DHD is the same in rats and mice. The hybridization conditions used to detect the rat homologue of DHD in rat brain tissue differed from those used to detect the transcript in mice only in that the stringency of the post-hybridization washes were reduced.

No hybridization was observed in the rat striatum using the post-hybridization washes employed following the in situ hybridization of mouse brain sections. However, when the stringency ofthe post-hybridization washes was lowered (2 x 60' in 1X SSC @
42oC, 2 x 60' in O.SX SSC @ 42oC, 2 x 60' in 0.25X SSC @ room temperature), the DHD
oligonucleotide probe specifically labelled the adult rat striatum, nucleus accumbens and olfactory tubercule in a pattern indistinguishable from that observed in mouse brain sections. It appears, therefore, that a transcript which shares nucleotide sequence and expression pattern is present in both mice and rats. The evolutionary conservation of DHD suggests that it is important for normal fiznction of the basal ganglia.
The mechanism by which only a small group of neurons in the striatum and cortex are rendered vulnerable to this ubiquitously expressed mutant protein is not known. The present inventors hypothesize that the presence of the expanded polyglutamine tract in huntingtin alters gene expression in the striatum.
EXAMPLE 8 - Analysis of :DHD in Genomic DNA
Genomic DNA was isolated from wild-type and HD mice and subjected to Southern blot analysis using pDHD as a hybridization probe. Analysis of the size of the fragments that hybridized with pDHD demonstrated that there was no difference in the size of the hybridizing fragments between the wild-type and HD mice. Figure 5 shows the genomic DNA
restriction fragments that hybridized with pDHD were the same in wild-type and HD mice.
The size of the hybridizing BamHI and EcoIRI fragment s in each genomic DNA sample is approximately 8 kb and 3 kb, respectively. This Southern blot analysis indicates that the gene encoding DHD

is present as a single-copy in the mouse genome. The numbers at the left of the blot are the relative mobility of molecular weight markers ( 1 kb ladder, BioRad).
The size of the hybridizing f~amHI and EcoRI fragments in each genomic DNA
sample is approximately 7 kb and 3 kb, respectively. The fact that only one genomic DNA
fragment hybridized with the DHD probe indicates that a single copy of the gene is present in the mouse genome.
EXAMPLE 9 - Isolation and Characterization of cDNA DHD
In order to isolate DHD cDNA clones, oligonucleotide probes 1 and 2 were used in 5' and 3' Rapid Amplification of cDNA Ends (RACE) reactions using commercially, prepared RACE-ready mouse striatal cDNA (Clontech). Several independent clones were isolated and those that contained the sequence of pDHD were selected for further analysis. Each of the S' RACE
clones was identical in sequence over the length that the clones could be aligned. The difference in length between these clones is a result of termination of the original reverse-tra.nscriptase reaction at different positions along the mRNA. No difference in size or sequence was detected between several 3' RACE clones. The longest S' RACE
clone and one 3' RACE clone were completely sequenced using internal primers. The present inventors were able to isolate a very short clone that extended the 5' RACE clone using an internal primer (probe 3, 5'- CTA TTT CAC AAG AGA CTG ACC AGC CAA TAA ATC TC- 3') The compiled sequence of the first DHD cDNA clone, named cDHD-1 is presented in Fig. 6.
cDHD-1 is 3235 by in length. The restriction map of cDHD-1 is shown in Fig. 7.

The mRNA that hybridized with pDHD was approximately 7.5 kilobases in length.
In order to obtain DHD cDNA clone that was larger than cDHD-1, the present inventors screened a mouse brain cDNA library.Several clones were identified that hybridized with the pDHD
probe. The sequence of the largest of these cDNA clones, cDHD-2, was determined. The sequence (Fig. 8) was 5753 base pairs in length. The restriction map of cDHD-2 is shown in Fig. 9.
cDHD-1 and cDHD-2 share sequence identity over 2095 bp. However, the 5' 1142 by of cDHD-1 and the 5' 1689 by of cDHD-2 are unique to each clone. Clone cDHD-2extends 1969 by in the 3' direction compared to cDHD-1. A schematic showing the regions of sequence identity and the unique sequences of cDHD-1 and -2 are shown in Fig.
10.

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SEQUENCE LISTING
<110> ROBERTSON, Harold DENOVAN-WRIGHT, Eileen NOVANEURON, INC.
<120> GENE NECESSARY FOR STRIATAL FUNCTION AND tJSES THEREFOR
<130> 36541-0001 <140>
<141>
<160> 10 <170> PatentIn Ver. 2.0 <210> 1 <211> 3236 <212> DNA
<213> mouse <400> 1 cactgaagct ggtccacgtc tataaacagg tgacactggc tgcagcaaaa agccattcga 60 tccacacaaa ttgatcttct atcatcttgg aatctgaatt gcagggagga gcagtatgta 120 agacgaccgt ttaattcagg cattccgaag gcatgagcgc atggattctg tcaccaagcg 180 tataaaagga ccctggcatt gggaaaccta tgacggactg tttt~tgctgt agaagtaggg 240 attttacaga agtctccttg aatttgccct gcctggggca gttttgcaga ggaacctgcc 300 agagatttat tggctggtca gtctcttgtg aaatagtatc atgt=gagaaa cagtttgtag 360 aaaaaaacta tacctgggaa gacctttgca acattgttcc ttccatgggc caagactcag 420 ttaggaggca taaatctgcc cggaataaac taggccagga tacagccatg tttagttaat 480 aatttggttt tagaattcac acaggcagga ttggtttttt tgtgtcttgg caagtggagc 540 atatttaaca tacaggcatg ggaatcctgc ctcttagctt ttc<:caccct cttgtctcac 600-caagtttttt ctctccaaag gtttccagga atttctcatt aatggctgat gcaaacttag 660 tgaataataa tgaatataaa caatgctcac ctcaccaaaa ttat:attatt tgcagtcatt 720 tgtgataaca caaattttat cgcaatggtt attatttaat ttgt:ggccac acactgtggt 780 tatcttttgt tgtggttgtt tctgagaaaa tgttcttgga tatgtaagtg ccaataccag 840 tgtgaagtat tgatcccggg cagcaaaata cagcctaagg tttgtaaaca tcaattctat 900 ctcagttcat cagagggcct gagaagctgc ggggcagtgt aaagtaaagt atgctgggct 960 ggtggtggtc agcctcccgc ctgaagagtg accagtgctg gcccgacgga tcgctgagat 1020 attctcccat aatggcaaaa aaataggcag tttgatgtga cctc3tttagt gtggctctcc 1080 tcttttgagc atgtgttagc atttttattt tatactcatc cagt;gaactc tgctcttcca 1140 agtgtgttca tgtatgtgct agatatatta gcacagcctg ccttctgctg cacaacgcct 1200 tagagacccg gcctttcaat gagcttagct tgtgctctgt ttctgctctc ttaggtctaa 1260 actatggtgt cagttttaat agaacaaaag tatgcatctt gccttggctt gagccttttc 1320 gttttcaatg ctgacttctc ccctttctct cctgtgctca cctt:accttt ccagagtgta 1380 agggacaact tttaaggagg cgtgtccctg gtaggggcat ccct=gttcac caggtgcctg 1440 tcatcacccc acttgactga catctaccct ggtgactatg ggttcctctt gtttgtaggg 1500 aacggtggct ccaggtggag gcatcaatct gttgggttct ggttcccggc tgcctttggt 1560 tttgaaagtc tcttctctgt atattcctac cctgcatttg ctttgtgtgg tgctgatgct 1620 gtgcgcagta ggattcttgg atgactctcc atcagtcaca gactccccct gttgcaaagt 1680 gtcaggctga ctcgacagtc accgtaaaat ctgagtcagt cacacacagg ctgtcagcca 1740 cggcttccac ttgcatggct attctatttt cacacgtgag tttctgt:tgc tggctggctg 1800 actggcatta tctatgctaa gttgaaatca ggagtgccca gcagag<:cca tcattctcac 1860 tgtctttgaa acaaagctgt acggtttgat cgatgaacgt atttaaagca tttcatgcaa 1920 tgacaaagtg ctcagtagtg gaaggcaggc tgtgaccagt ctgcctgctc cttactataa 1980 ttgtgaggat ttgttactgg aacagtacat ggaggcctga ccttgtgggg gcacagggtg 2040 gaaccttagc tgaatatagt gtgtgtctca agaggaagtc agggtactag ctcagtgctc 2100 aatctccagg tactatatat acatttgccc gttttatctc taatgtgaaa taaatcccca 2160 aacacttgtt tatcgtgtag cgtacctaaa agactattct attatgggtg tccccacttt 2220 cttggtttgg tcaccccgat cccccggtct tctgctgtat ctagaacagt gactataaat 2280 gatgtatggg aatagtgttt ccatatgatc tgttgtctgg agt.atatgct acatgttcaa 2340 ttactgtaca aaaacccagt gcagctgatg atgcaaagca gtctctctct gtgtacagtg 2400 ccccacctat ttaaaaatca cgtacaascc cagaacactg tgaaacactt aacataagaa 2460 caaacgcagc gtctggattc tttccaagga gagcagcttt ctccacagga acacagtaac 2520 aaaagaggtc cgccgccatc cacacccagc caagacacct cagaggccat agggacaacc 2580 tccttgctgg ccaacacctg ctggagcagg ggcacaggtc ccagcaactg atcctcagtg 2640 gatgggtccg cagtcaaagc cttaatgggc tctcttttga aggggaaaga aagaatttca 2700 agcttatgat atccaacatt attatagttg atgagttagt aaattccaaa aaaaaaagat 2760 gattttatat gtatgacata aaaaaaatct ttgtaaagtg cgc:aagtgca ataatttaaa 2820 gaggtcttat ctttgcattt ataaattata aatattgtac atcttgtgtaa tttttcatgt 2880 attcatttgc agtctttgta tttaaaaaaa ctttactgtt atgtttgtat aatagaacat 2940 taatcattta ttataactca gacaaggtgt aaataaattc ataattcaaa cagccagtat 3000 atatgcatat atgggtgtta cattgcaaaa atctctatct ttgttctatt cacatgctta 3060 aagaagtaag aaatcttttg tggatatgta attatacata taaagtatat atatatgtat 3120 gatacatgaa atatatttag aaatgttcat aattttaatg gatatt~~ttt ggtgtgaata 3180 attgaataca acatttttaa aatgaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaa 3236 <210> 2 <211> 5752 <212> DNA
<213> mouse <400> 2 aagtgtaaat aaaataaaca tctaataaaa aaaattacat accatagagg aacaagataa 60 tttctgccca acttcatacc ctccagcgta tagtgttgag gtt:tggtctg ttgctgtgta 120 ttgtaatgta atgttaaatt ctctacctga aggtctaggc ctacaagtga attctcatgt 180 ttatagagtt ttgttgtgca aaccttgttc cttaatttaa aactatggtt aaaaaacaaa 240 acaaaactgg ctacagccaa taactgaagg gggttacctt gttgaagggg tggaaaagag 300 agaggaggaa gaagggagtt caagagaagg agaagaacaa gaggagagga ggaagctgcc 360 acgaggggag atgggccatg agaacttggc caggagaaat agccagtatc tggagtacac 420 cactgaggag gtagccaggc tagcagttag aagagtagat taggggttat ttttccccca 480 ctccacatag ttatcaaagc caaataaaat aaccatagtc tg<~gtctcat ctatttgtaa 540 gctagttggg tataagatta atttggctgt actacagttt agatttctaa cataggaact 600 atcaaaaact tgctcaaaca agaacatgct gacaatattt taaaatgatt atttatattg 660 tttgcacttt ctaaagtttc ttctaaatgt tccatggtca aattaaaaaa tatacatatt 720 ggctattaaa ttcgtctaag 'tggggctgga gagatagctc agaggttaag agcactgact 780 gctcttccag aggtcctgag ttcaattccc agcgaccaca tggtggctca cagccatctg 840 taatagatag gatctgacgc ~~ctcttctgg agtgtctgaa gacagctaca atgtactcat 900 atatattaaa taaataatat tagaaaattc ttctaagtgt atcatttata gaatatttaa 960 tatataaagt aaatgcctca ggaaatataa acttggaatt aaat:caaaga acttcatgag 1020 tagtgggcca caaaaaatgt gtaccagggg aagaccggag ggaggggaga aggaagggat 1080 ggagatagaa ttttgcctct ~3cattccttg ggctggcaca ggtataatgc tgtgggaatt 1140 gggaaactac aaggaagctg caaagctggg cggaactcgt ttcc:gcaagc tgggctcatc 1200 taagtgtcca tgcatggctg ccacactgca gtgaacttta aaac:atttgt gttccagaga 1260 tgtagagatg ctcacaatag tacaaaggcg ggagggaggt atttccagac taagaggaag 1320 aaaaaccatt gctgattaaa catctgcata tgagcgcccc cacca ccata cacacacaca 1380 cacacacaca cacacacaca caaccaaaca gaacaaatac acatgcatgt ctacagcctg 1440 caggaacaaa atggtatgtc tgtgaggaac caggagatgc acaggtccta acctctgtct 1500 cctacaagcc ctgaagtctg gtcagggtca aatgtacaaa agcaggctaa ggaagctgtt 1560 tagtgaaaga tttttttctt caactctagg aacaacctat ttcctaggat ttggagagtg 1620 ctcaggagga aacattcaga caactgatgc tctctgtgta ccc<~agattc aggtattggg 1680 gtagttagtt gtgctcatgt atgtgctaga tatattagca cagcctgcct tctgctgcac 1740 aacgccttag agacccggcc tttcaatgag cttagcttgt gctctgtttc tgctctctta 1800 ggtctaaact atggtgtcag ttttaataga acaaaagtat gcatcttgcc ttggcttgag 1860 ccttttcgtt ttcaatgctg acttctcccc tttctctcct gtgctcacct tacctttcca 1920 gagtgtaagg gacaactttt aaggaggcgt gtccctggta ggg~3catccc tgttcaccag 1980 gtgcctgtca tcaccccact tgactgacat ctaccctggt gactatgggt tcctcttgtt 2040 tgtagggaac ggtggctcca ggtggaggca t:caatctgtt gggttctggt tcccggctgc 2100 ctttggtttt gaaagtctct tctctgtata ttcctaccct gcatttgctt tgtgtggtgc 2160 tgatgctgtg cgcagcagga ttcttggatg actctccatc agtcacagac tccccctgtt 2220 gcaaagtgtc aggctgactc gacagtcacc gtaaaatctg agtcagtcac acacaggctg 2280 tcagccacgg cttccacttg catggctatt ctattttcac acgtgagttt ctgttgctgg 2340 ctggctgact ggcattatct atgctaagtt gaaatcaggg gtgcccagca gagcccatca 2400 ttctcactgt ctttgaaaca aagctgtacg gtttgatcga tgaacgt.att taaagcattt 2460 catgcaatga caaagtgctc agtagtggaa ggcaggctgt gaccagtctg cctgctcctt 2520 actataattg tgaggatttg ttactggaac agtacatgga ggcctgacct tgtgggggca 2580 cagggtggaa ccttagctga atatagtgtg tgtctcaaga ggaagtcagg gtactagctc 2640 agtgctcaat ctccaggtac tatatataca tttgcccgtt ttatctctaa tgtgaaataa 2700 atccccaaac acttgtttat cgtgtagcgt acctaaaaga ctattct:att atgggtgtcc 2760 ccactttctt ggtttggtca ccccgatccc ccggtcttct gctgtatcta gaacagtgac 2820 tataaatgat gtatgggaat agtgtttcca tatgatctgt tgtctggagt atatgctaca 2880 tgttcattta ctgtacaaaa acccagtgca gctgatgatg caaagcagtc tctctctgtg 2940 tacagtgccc -cacctattta aaaatcacgt acttgcccag aacactqtga aacacttaac 3000 ataagaacaa acgcagcgtc tggattcttt ccaaggagag cagcttt:ctc cacaggaaca 3060 cagtaacaaa agaggtccgc cgccatccac acccagccaa gacacct:cag aggccatagg 3120 gacaacctcc ttgctggcca acacctgctg gagcaggggc acaggt<:cca gcaactgatc 3180 ctcagtggat gggtctgcag ccaaagcctt aatgggctct cttttgaagg ggaaagaaag 3240 aatttcaagc ttatgatatc caatattatt atagttgatg agttagt:aaa ttccaaaaaa 3300 aaaagatgat tttatatgta tgacataaaa aaaatctttg taaagtgcgc aagtgcaata 3360 atttaaagag gtcttatctt tgcatttata aattataaat attgta<:atg tgtgtaattt 3420 ttcatgtatt catttgcagt ctttgtattt aaaaaaactt tactgttatg tttgtataat 3480 agaacattaa tcatttatta taactcagac aaggtgtaaa taaattcata attcaaacag 3540 ccagtatata tgcatatatg ggtgttacat tgcaaaaatc tct.atctttg ttctattcac 3600 atgcttaaag aagtaagaaa tcttttgtgg atatgtaatt atacatataa agtatatata 3660 tatgtatgat acatgaaata tatttagaaa tgttcataat tttaatggat attctttggt 3720 gtgaataatt gaatacaaca tttttaaaat aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3780 aaaatttttt tttttttttt ~~tattccaga gattaaagac actagatctt taaccttgaa 3840 gggcaggcaa gaggtcggca ~3tgctgtcaa catagaagtc agggaccatt ttcttcttga 3900 acatgcagtc actttcctga ttgctcttca catcctcaag gctccggaat tccgggggtg 3960 tggtgggctt tgatctcagg <3ctctggagg cagaagcagg cagatctctg tgaatatgag 4020 gccagcctgc actacacaga gctccagacc agtcatggct acat:catgaa accctgtctc 4080 aaaaagaaaa taaaaactgt t:gtgtttcta ccatagtgtt aaactcagag tctgagtaat 4140 gtcgggctga catgctcggg t:gtttaacat accttcagct ttgacgaggc gctgaacagt 4200 caaagtctgg ccttggggag c:ggtggctgt gtttgtgctc aagtccaccg tgaaatcctg 4260 attgtgaatt tggacaaccg t:gtccttctt cttggccttc catgcaacct ccaacttcat 4320 gttggtcatt ttgtcaaaac actgtgtgat gtttttatca atatactgcc attccacata 4380 tgtagagatg tagtctgcct dgctttcctt ttctttagcc aatcgaatgc tcttgatcat 4440 gccctcaatc tcatctctag catttatcac gtctctgcta attcctgaaa cttgaatcga 4500 agttttcttc tggttcatct c:aatggtgat gttcagttcc ttctgaatct cattcagttt 4560 ctcgtactcc tccatgtcaa agtcactgac acactcatcg tcattggtgt aggaaagctg 4620 ctctttggta atcagttcct t:tagccagga gattgttttg ttcacactgt ctacccctga 4680 accacatacc tggaaaactg t:gtgctctat tttcttttcc aaaaccaggg tgttcttttt 4740 gggggaagct tgcttgggaa agccaagaaa ggctaaagag aaaatggaaa ttaatgtttc 4800 ttttactccc ttcaacatca aggttaggaa tatgtatttc ataaaag<aa acaactcaca 4860 ggcaatctta gacatcactg actgcttggc aggcgactgc ttggggggag ctggagagcc 4920 ttctcttt.ct ttcatgttgt c:gtaaaaaaa ttgcagaata tggggctgga agataacaac 4980 tttaactctc ttcacagcct c~cactgattt tttctggaca aattcttcaa tggcatctat 5040 tatcgctttt gctactacgt t.tgggtcctg ttgagcattt ccttcaaaaa caaaaaaagc 5100 acatttttaa aaagtcaagg t.taagatcca cctgcaaaaa aaagctgcaa tataagcgag 5160 gaattctagt tgtcacagga a.ataaaaatg tctgttccca ctataatcaa tgtagactga 5220 taatattatg ccagcaaata a~ttttgaagt cctaggcaca gtgggaggag gttttgttcc 5280 acgctgttca taagccaata ccccagcaaa agaccttaaa ggacaacttg taatttggga 5340 cattcacatc tgtcctcttc atctgatctg gctcccagtg tcactctca a acacggtcct 5400 tagagggaca atttatccct gcctctgctt gatcttatgc atgtatctgt attcttccag 5460 ccatccctgg cgacctgatt tttctaaggc acccaaaact gtaagctact tcttataatc 5520 tataattctg agcatattag ttagcctgag cctccaggat atctttcttc cctatactca 5580 gtccagtttt agctgcccag aaggattcaa agctgatcta cgagtagatc actcctgtct 5640 acagcttgtt ccagatcttg tttctcaagc cctggaagcc atcagccagg taagattgta 5700 aaacaatccc tttctaatca tgggtgtggc ccaaagtgaa tggccggaat tc 5752 <210> 3 <211> 475 <212> DNA
<213> mouse <400> 3 tgtatgggaa tagtgtttcc atatgatctg ttgtctggag tatatgctac atgttcattt 60 actgtacaaa aacccagtgc agctgatgat gcaaagcagt ctctctctgt gtacagtgcc 120 ccacctattt aaaaatcacg tacttgccca gaacactgtg aaacacttaa cataagaaca 180 aacgcagcgt ctggattctt tccaaggaga gcagctttct ccacaggaac acagtaacaa 240 aagaggtccg ccgccatcca cacccagcca agacacctca gaggccatag ggacaacctc 300 cttgctggcc aacacctgct ggagcagggg cacaggtccc agcaactgat cctcagtgga 360 tgggtctgca gccaaagcct taatgggctc tcttttgaag gggaaagaaa gaatttcaag 420 cttatgatat ccaatattat tatagttgat gagttagtaa attccaaaaa aaaaa 475 <210> 4 <211> 20 <212> DNA
<213> Artificial Sequence <220>
<223> Description of P,rtificial Sequence: primer <400> 4 agggctgtca atcatgctgg 20 <210> 5 <211> 20 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: primer <400> 5 aaactcacgg tcggtgcagc 20 <210> 6 <211> 24 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: probe <400> 6 attaaccctc actaaatgct gtat 24 <210> 7 <211> 30 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: probe <400> 7 cattatgctg agtgatatct ttttttttcg 30 <210> 8 <211> 38 <212> DNA, <213> Artificial Sequence <220>
<223> Description of F.rtificial Sequence: probe <400> 8 gaacatgtag catatactcc agacaacaga tcatatgg 3g <210> 9 <211> 32 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: probe <400> 9 cagcttctcc acaggaacac agtaacaaag ag 32 <210> 10 <211> 35 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: primer <400> 10 ctatttcaca agagactgac cagccaataa atctc 35

Claims (24)

1. An isolated polynucleotide segment, comprising a polynucleotide sequence, wherein the polynucleotide sequence is selected from the group consisting of:
(a) a sequence comprising SEQ ID NO:1;
(b) a sequence comprising SEQ ID NO:2;
(c) a sequence having nucleotides 1140 to 3235 of SEQ ID NO:1;
(d) a sequence which is at least 80% homologous with a sequence of (a), (b) or (c);
(e) variants of (a), (b), (c) or (d), and;
(f) a sequence which hybridizes to (a), (b), (c) or (d) under stringent conditions.

2. The isolated polynucleotide segment of claim 1, wherein the isolated polynucleotide segment is RNA.

3. A vector comprising the isolated polynucleotide segment of claim 1.

4. An isolated host cell comprising the vector of claim 3.

5. A process for producing a polypeptide of a polynucleotide sequence of claim 1 comprising the step of culturing the host cell of claim 4 under conditions sufficient for the production of said polypeptide.

6. An isolated polynucleotide fragment, comprising a polynucleotide sequence, wherein the polynucleotide sequence is selected from the group consisting of:
(a) a sequence having at least 15 sequential bases of nucleotides 1140 to 3235 of SEQ ID NO:
1;
(b) a sequence having at least 30 sequential bases of nucleotides 1140 to 3235 of SEQ ID NO:
1;
(c) a sequence having at least 50 sequential bases of SEQ ID NO:1 or SEQ ID
NO: 2;
(d) a sequence which is at least 90% homologous with a sequence of (a), (b) or (c);

(e) variants of (a), (b), (c) or (d), and;
(f) a sequence which hybridizes to (a), (b), (c) or (d) under stringent conditions.

7. A polynucleotide segment of claim 6, wherein the polynucleotide fragment is RNA.

8. A vector comprising the isolated polynucleotide fragment of claim 6.

9. An isolated host cell comprising the vector of claim 8.

10. A method for identifying a compound which inhibits or promotes the activity of a polynucleotide segment of claim 1, comprising the steps of:
(a) selecting a control animal having said segment and a test animal having said segment;
(b) treating said test animal using a compound; and, (c) determining the relative quantity of RNA corresponding to said segment, as between said animals.

11. A method of claim 10, wherein said animal is a mammal.

12. A method of claim 11, wherein said mammal is a mouse.

13. A method of claim 12, wherein said mouse is R6/2 transgenic mouse.

14. A method for identifying a compound which inhibits or promotes the activity of a polynucleotide segment of claim 1, comprising the steps of:
(a) selecting a host cell of claim 4;
(b) cloning said host cell and separating said clones into a test group and a control group;
(c) treating said test group using a compound; and (c) determining the relative quantity of RNA corresponding to said segment, as between said test group and said control group.

15. An isolated mouse polypeptide having a nucleotide sequence as set forth in claim 1.

16. A vector which comprises the polypeptide of claim 15.

17. A host cell which is transformed with the vector of claim 16.

18. A method for diagnosing the presence of or the predisposition for a CAG
repeat disorder, said method comprising determining the level of expression of RNA
corresponding to the segments of claim 1 in an individual relative to a predetermined control level of expression, wherein a decreased expression of said RNA as compared to said control is indicative of a CAG repeat disorder.

19. The method of claim 18 wherein said expression is measured by in situ hybridization.

20. The method of claim 18 wherein said expression is measured by fluorescent in situ hybridization.

21. The method of claim 18 wherein said expression is measured using a polymerase chain reaction.

22. The method of claim 18 wherein said expression is measured using a DNA
fingerprinting technique.

23. The method of claim 18 wherein said CAG repeat disorder is Huntington's disease.

24. An isolated polynucleotide segment, comprising a polynucleotide sequence which retains substantially the same biological function or activity as the polynucleotide encoded by the polynucleotide sequence of claim 1.