EP0804226A1 - Human choline acetyltransferase - Google Patents

Abstract

Human choline acetyltransferase polypeptide and DNA (RNA) encoding such polypeptide and a procedure for producing such polypeptide by recombinant techniques is disclosed. Also disclosed are methods for utilizing such polypeptide for the treatment of cognitive and neurological deficiencies or mental disturbances such as degenerative nervous system disorders, for example, Alzheimer's Disease, ALS and other cholinergic defects, and antagonists for treating Parkinson's Disease and other disorders relating to an over-expression of acetylcholine. Also disclosed are diagnostic methods for detecting a mutation in the human Choline Acetyltransferase nucleic acid sequence.

Description

HUMAN CHOLINE ACETYLTRANSFERASE

This invention relates to newly identified polynucleotides, polypeptides encoded by such polynucleotides, the use of such polynucleotides and polypeptides, as well as the production of such polynucleotides and polypeptides. More particularly, the polypeptide of the present invention is choline acetyltransferase, sometimes hereinafter referred to as "hChAT" . The invention also relates to inhibiting the action of such polypeptides.

A human choline acetyltransferase gene has been isolated from the human brain (McGeer P.L. et al., Life Sci., 34:2319- 2338 (1984)). Choline acetyltransferase is specifically expressed in cholinergic neurons. Choline acetyltransferase is an enzyme which catalyzes a reaction which yields the neurotransmitter acetylcholine. Although choline acetyltransferase expression has been found in both neurons and certain non-neuronal tissues, such as placenta (Schuberth, J., Biochim. Biophys. Acta, 122:470-481 (1966)) and spermatozoa (Ibanez, C.F. and Persson, H. , Eur. J. Neurosci., 3:1309-1315 (1991)), the expression of this enzyme is largely limited to certain neurons.

The 5' flanking region of the human choline acetyltransferase gene differs from the rodent gene in that the rodent gene has a "TATAA" box consensus sequence upstream of the transcription start site, but no such element is found in the human gene. The rodent gene also differs in that it contains at least three promoters and the sequence corresponding to only one of the promoters, the M type, is found in the human gene (Hersh, L.B., et al., J. Neurochem. , 61:306-314 (1993) ) .

The control of motor behavior constitutes one of the most important functions of the central nervous system. Numerous regions of the brain are involved in this process that is integrated ultimately in the motor neurons of the spinal cord, the "final common path" in the control of movement. These neurons, which lie in the ventral horn, exhibit a cholinergic phenotype and, therefore, express choline acetyltransferase. Choline acetyltransferase is a specific marker of the cholinergic system.

In accordance with one aspect of the present invention, there is provided a novel mature polypeptide which is hChaT, as well as biologically active and diagnostically or therapeutically useful fragments, analogs and derivatives thereof.

In accordance with another aspect of the present invention, there are provided isolated nucleic acid molecules encoding hChaT, including mRNAs, DNAs, cDNAs, genomic DNA as well as biologically active and diagnostically or therapeutically useful fragments, analogs and derivatives thereof.

In accordance with yet a further aspect of the present invention, there is provided a process for producing such polypeptide by recombinant techniques which comprises culturing recombinant prokaryotic and/or eukaryotic host cells, containing a hChaT nucleic acid sequence, under conditions promoting expression of said protein and subsequent recovery of said protein. In accordance with yet a further aspect of the present invention, there is provided a process for utilizing such polypeptides, or polynucleotide encoding such polypeptide for therapeutic purposes, for example, to treat amyotrophic lateral sclerosis (ALS), Alzheimer's Disease, familial disautonomia, Huntington's Disease, mental retardation, memory loss, myasthenia gravis and other disorders known to involve the cholinergic system or affect its pathways and nerves in the body.

In accordance with another aspect of the present invention there are provided nucleic acid probes comprising nucleic acid molecules of sufficient length to specifically hybridize to hChaT sequences.

In accordance with yet a further aspect of the present invention, there are provided antibodies against such polypeptides.

In accordance with another aspect of the present invention there is provided a method of diagnosing a disease or a susceptibility to a disease, related to a mutation in hChaT nucleic acid sequences and the protein encoded by such nucleic acid sequences, for example, Alzheimer's disease.

In accordance with yet another aspect of the present invention, there are provided antagonists to such polypeptides, which may be used to inhibit the action of such polypeptides, for example, in the treatment of Parkinson's Disease and accidental overdoses with various pharmaceuticals and contamination by toxins.

These and other aspects of the present invention should be apparent to those skilled in the art from the teachings herein.

The following drawings are illustrative of embodiments of the invention and are not meant to limit the scope of the invention as encompassed by the claims.

Figure l depicts the cDNA sequence and corresponding deduced amino acid sequence of hChat. The hChat polypeptide as shown is the putative mature polypeptide. The standard one-letter abbreviations for amino acids are used.

In accordance with an aspect of the present invention, there is provided an isolated nucleic acid (polynucleotide) which encodes for the mature polypeptide having the deduced amino acid sequence of Figure 1 or for the mature polypeptide encoded by the cDNA of the clone deposited as ATCC Deposit No. 75856 on August 9, 1994.

A polynucleotide encoding a polypeptide of the present invention may be obtained from fetal liver and fetal lung. The polynucleotide of this invention was discovered in a cDNA library derived from human fetal lung. It contains an open reading frame encoding a protein of 677 amino acid residues. The protein exhibits the highest degree of homology to pig choline acetyltransferase with 43 % identity and 64 % similarity over a 145 amino acid stretch.

The polynucleotide of the present invention may be in the form of RNA or in the form of DNA, which DNA includes cDNA, genomic DNA, and synthetic DNA. The DNA may be double- stranded or single-stranded, and if single stranded may be the coding strand or non-coding (anti-sense) strand. The coding sequence which encodes the mature polypeptide may be identical to the coding sequence shown in Figure 1 or that of the deposited clone or may be a different coding sequence which coding sequence, as a result of the redundancy or degeneracy of the genetic code, encodes the same mature polypeptide as the DNA of Figure 1 or the deposited cDNA.

The polynucleotide which encodes for the mature polypeptide of Figure 1 or for the mature polypeptide encoded by the deposited cDNA may include: only the coding sequence for the mature polypeptide; the coding sequence for the mature polypeptide and additional coding sequence such as a leader or secretory sequence or a proprotein sequence; the coding sequence for the mature polypeptide (and optionally additional coding sequence) and non-coding sequence, such as introns or non-coding sequence 5' and/or 3' of the coding sequence for the mature polypeptide.

Thus, the term "polynucleotide encoding a polypeptide" encompasses a polynucleotide which includes only coding sequence for the polypeptide as well as a polynucleotide which includes additional coding and/or non-coding sequence.

The present invention further relates to variants of the hereinabove described polynucleotides which encode for fragments, analogs and derivatives of the polypeptide having the deduced amino acid sequence of Figure 1 or the polypeptide encoded by the cDNA of the deposited clone. The variant of the polynucleotide may be a naturally occurring allelic variant of the polynucleotide or a non-naturally occurring variant of the polynucleotide.

Thus, the present invention includes polynucleotides encoding the same mature polypeptide as shown in Figure 1 or the same mature polypeptide encoded by the cDNA of the deposited clone as well as variants of such polynucleotides which variants encode for a fragment, derivative or analog of the polypeptide of Figure 1 or the polypeptide encoded by the cDNA of the deposited clone. Such nucleotide variants include deletion variants, substitution variants and addition or insertion variants.

As hereinabove indicated, the polynucleotide may have a coding sequence which is a naturally occurring allelic variant of the coding sequence shown in Figure 1 or of the coding sequence of the deposited clone. As known in the art, an allelic variant is an alternate form of a polynucleotide sequence which may have a substitution, deletion or addition of one or more nucleotides, which does not substantially alter the function of the encoded polypeptide.

The polynucleotides of the present invention may also have the coding sequence fused in frame to a marker sequence which allows for purification of the polypeptide of the present invention. The marker sequence may be a hexa- hiεtidine tag supplied by a pQE-9 vector to provide for purification of the mature polypeptide fused to the marker in the case of a bacterial host, or, for example, the marker sequence may be a hemagglutinin (HA) tag when a mammalian host, e.g. COS-7 cells, is used. The HA tag corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson, I., et al., Cell, 37:767 (1984)).

The present invention further relates to polynucleotides which hybridize to the hereinabove-described sequences if there is at least 50% and preferably 70% identity between the sequences. The present invention particularly relates to polynucleotides which hybridize under stringent conditions to the hereinabove-described polynucleotides . As herein used, the term "stringent conditions" means hybridization will occur only if there is at least 95% and preferably at least 97% identity between the sequences. The polynucleotides which hybridize to the hereinabove described polynucleotides in a preferred embodiment encode polypeptides which retain substantially the same biological function or activity as the mature polypeptide encoded by the cDNA of Figure l or the deposited cDNA.

The deposit(s) referred to herein will be maintained under the terms of the Budapest Treaty on the International Recognition of the Deposit of Micro-organisms for purposes of Patent Procedure. These deposits are provided merely as convenience to those of skill in the art and are not an admission that a deposit is required under 35 U.S.C. §112. The sequence of the polynucleotides contained in the deposited materials, as well as the amino acid sequence of the polypeptides encoded thereby, are incorporated herein by reference and are controlling in the event of any conflict with any description of sequences herein. A license may be required to make, use or sell the deposited materials, and no such license is hereby granted. The present invention further relates to a hChat polypeptide which has the deduced amino acid sequence of Figure 1 or which has the amino acid sequence encoded by the deposited cDNA, as well as fragments, analogs and derivatives of such polypeptide.

The terms "fragment," "derivative" and "analog" when referring to the polypeptide of Figure 1 or that encoded by the deposited cDNA, means a polypeptide which retains essentially the same biological function or activity as such polypeptide. Thus, an analog includes a proprotein which can be activated by cleavage of the proprotein portion to produce an active mature polypeptide.

The polypeptide of the present invention may be a recombinant polypeptide, a natural polypeptide or a synthetic polypeptide, preferably a recombinant polypeptide.

The fragment, derivative or analog of the polypeptide of Figure 1 or that encoded by the deposited cDNA may be (i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, or (ii) one in which one or more of the amino acid residues includes a substituent group, or (iii) one in which the mature polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol) . Such fragments, derivatives and analogs are deemed to be within the scope of those skilled in the art from the teachings herein.

The polypeptides and polynucleotides of the present invention are preferably provided in an isolated form, and preferably are purified to homogeneity.

The term "isolated" means that the material is removed from its original environment (e.g., the natural environment if it is naturally occurring) . For example, a naturally- occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated. Such polynucleotides could be part of a vector and/or such polynucleotides or polypeptides could be part of a composition, and still be isolated in that such vector or composition is not part of its natural environment.

The present invention also relates to vectors which include polynucleotides of the present invention, host cells which are genetically engineered with vectors of the invention and the production of polypeptides of the invention by recombinant techniques.

Host cells are genetically engineered (transduced or transformed or transfected) with the vectors of this invention which may be, for example, a cloning vector or an expression vector. The vector may be, for example, in the form of a plasmid, a viral particle, a phage, etc. The engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants or amplifying the hChat genes. The culture conditions, such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.

The polynucleotides of the present invention may be employed for producing polypeptides by recombinant techniques. Thus, for example, the polynucleotide may be included in any one of a variety of expression vectors for expressing a polypeptide. Such vectors include chromosomal, nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40; bacterial plasmids; phage DNA; baculovirus; yeast plasmids; vectors derived from combinations of plasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl pox virus, and pseudorabies. However, any other vector may be used as long as it is replicable and viable in the host.

The appropriate DNA sequence may be inserted into the vector by a variety of procedures. In general, the DNA sequence is inserted into an appropriate restriction endonuclease site(s) by procedures known in the art. Such procedures and others are deemed to be within the scope of those skilled in the art.

The DNA sequence in the expression vector is operatively linked to an appropriate expression control sequence(s) (promoter) to direct mRNA synthesis. As representative examples of such promoters, there may be mentioned: LTR or SV40 promoter, the E. coli. lac or trp, the phage lambda P_L promoter and other promoters known to control expression of genes in prokaryotic or eukaryotic cells or their viruses. The expression vector also contains a ribosome binding site for translation initiation and a transcription terminator. The vector may also include appropriate sequences for amplifying expression.

In addition, the expression vectors preferably contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or such as tetracycline or ampicillin resistance in E. coli.

The vector containing the appropriate DNA sequence as hereinabove described, as well as an appropriate promoter or control sequence, may be employed to transform an appropriate host to permit the host to express the protein.

As representative examples of appropriate hosts, there may be mentioned: bacterial cells, such as E. coli, Streptomyces, Salmonella tvphimurium; fungal cells, such as yeast; insect cells such as Drosophila S2 and ____; animal cells such as CHO, COS or Bowes melanoma; adenoviruses; plant cells, etc. The selection of an appropriate host is deemed to be within the scope of those skilled in the art from the teachings herein.

More particularly, the present invention also includes recombinant constructs comprising one or more of the sequences as broadly described above. The constructs comprise a vector, such as a plasmid or viral vector, into which a sequence of the invention has been inserted, in a forward or reverse orientation. In a preferred aspect of this embodiment, the construct further comprises regulatory sequences, including, for example, a promoter, operably linked to the sequence. Large numbers of suitable vectors and promoters are known to those of skill in the art, and are commercially available. The following vectors are provided by way of example. Bacterial: pQE70, pQE60, pQE-9 (Qiagen) , pbs, pDIO, phagescript, psiX174, pbluescript SK, pbsks, pNH8A, pNH16a, pNH18A, pNH46A (Stratagene) ; ptrc99a, pKK223- 3, pKK233-3, pDR540, pRIT5 (Pharmacia) . Eukaryotic: pWLNEO, pSV2CAT, pOG44, pXTl, pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia) . However, any other plasmid or vector may be used as long as they are replicable and viable in the host.

Promoter regions can be selected from any desired gene using CAT (chloramphenicol transferase) vectors or other vectors with selectable markers. Two appropriate vectors are PKK232-8 and PCM7. Particular named bacterial promoters include lad, lacZ, T3, T7, gpt, lambda P_R, P_L and trp. Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-I. Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art.

In a further embodiment, the present invention relates to host cells containing the above-described constructs. The host cell can be a higher eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell. Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE- Dextran mediated transfection, or electroporation. (Davis, L. , Dibner, M., Battey, I., Basic Methods in Molecular Biology, (1986) ) .

The constructs in host cells can be used in a conventional manner to produce the gene product encoded by the recombinant sequence. Alternatively, the polypeptides of the invention can be synthetically produced by conventional peptide synthesizers.

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 RNAs 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., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989), the disclosure of which is hereby incorporated by reference.

Transcription of the DNA encoding the polypeptides of the present invention by higher eukaryotes is increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp that act on a promoter to increase its transcription. Examples including the SV40 enhancer on the late side of the replication origin bp 100 to 270, a cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.

Generally, recombinant expression vectors will include origins of replication and selectable markers permitting transformation of the host cell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiae TRPl gene, and a promoter derived from a highly-expressed gene to direct transcription of a downstream structural sequence. Such promoters can be derived from operons encoding glycolytic enzymes such as 3-phosphoglycerate kinase (PGK) , or-factor, acid phosphatase, or heat shock proteins, among others. The heterologous structural sequence is assembled in appropriate phase with translation initiation and termination sequences. Optionally, the heterologous sequence can encode a fusion protein including an N-terminal identification peptide imparting desired characteristics, e.g., stabilization or simplified purification of expressed recombinant product.

Useful expression vectors for bacterial use are constructed by inserting a structural DNA sequence encoding a desired protein together with suitable translation initiation and termination signals in operable reading phase with a functional promoter. The vector will comprise one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and to, if desirable, provide amplification within the host. Suitable prokaryotic hosts for transformation include E. coli. Bacillus subtilis. Salmonella tvphimurium and various species within the genera Pseudomonaε, Streptomyceε, and Staphylococcus, although others may also be employed as a matter of choice.

As a representative but nonlimiting example, useful expression vectors for bacterial use can comprise a selectable marker and bacterial origin of replication derived from commercially available plasmids comprising genetic elements of the well known cloning vector pBR322 (ATCC 37017) . Such commercial vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM1 (Promega Biotec, Madison, WI, USA) . These pBR322 "backbone" sections are combined with an appropriate promoter and the structural sequence to be expressed.

Following transformation of a suitable host strain and growth of the host strain to an appropriate cell density, the selected promoter is induced by appropriate means (e.g., temperature shift or chemical induction) and cells are cultured for an additional period.

Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification.

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.

Various mammalian cell culture systems can also be employed to express recombinant protein. Examples of mammalian expression systems include the COS-7 lines of monkey kidney fibroblasts, described by Gluzman, Cell, 23:175 (1981) , and other cell lines capable of expressing a compatible vector, for example, the C127, 3T3, CHO, HeLa and BHK cell lines. Mammalian expression vectors will comprise an origin of replication, a suitable promoter and enhancer, and also any necessary ribosome binding sites, polyadenylation site, splice donor and acceptor sites, tranεcriptional termination sequences, and 5' flanking nontranscribed sequenceε. DNA sequences derived from the SV40 splice, and polyadenylation sites may be used to provide the required nontranscribed genetic elements.

The hChat polypeptides can be recovered and purified from recombinant cell cultureε by 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. Protein refolding steps can be used, as necessary, in completing configuration of the mature protein. Finally, high performance liquid chromatography (HPLC) can be employed for final purification steps. The polypeptides of the present invention may be a naturally purified product, or a product of chemical synthetic procedures, or produced by recombinant techniques from a prokaryotic or eukaryotic hoεt (for example, by bacterial, yeast, higher plant, insect and mammalian cells in culture) . Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or may be non-glycosylated. Polypeptides of the invention may also include an initial methionine amino acid residue.

The hChat polypeptideε of the preεent invention catalyze the transfer of the acyl-group to choline to yield acetylcholine. A deficiency of the neurotransmitter acetylcholine leads to cognitive and/or neurological deficiencies and/or mood or mental disturbances, such as suffering from degenerative nervous system disorders.

Accordingly, the hChat polypeptideε of the preεent invention may be employed to treat amyotrophic lateral sclerosis, Alzheimer's Diseaεe, senile-dementia, multi- infarct dementia, familial disautonomia, Huntington's Disease, mental retardation, memory loss and myasthenia gravis.

The hChat polypeptides may also be employed to treat disorders known to involve the cholinergic system or affect its pathways and nerves in the body. Exampleε of εuch diεorderε include gut and GI diεorderε, cord disorders, including movement, continence and senεation, stem disorders, including sleep, blood presεure, respiration, and balance, hypothalamus disorders, including temperature, respiration, and endocrine function, and limbic syεtem disorders, including schizophrenia, memory disorders, and dementia.

Fragments of the full length hChaT gene may be used as a hybridization probe for a cDNA library to isolate the full length gene and to isolate other genes which have a high sequence similarity to the gene or similar biological activity. Probes of this type can be, for example, between 20 and 2000 bases. Preferably, however, the probes have between 30 and 50 base pairs. The probe may also be used to identify a cDNA clone corresponding to a full length transcript and a genomic clone or clones that contain the complete hChaT gene including regulatory and promotor regions, exons, and introns. An example of a screen comprises isolating the coding region of the hChaT gene by using the known DNA sequence to synthesize an oligonucleotide probe. Labeled oligonucleotides having a sequence complementary to that of the gene of the present invention are used to screen a library of human cDNA, genomic DNA or mRNA to determine which members of the library the probe hybridizes to.

In accordance with yet a further aspect of the present invention, there is provided a process for utilizing such polypeptides, or polynucleotides encoding such polypeptides, for in vitro purposes related to scientific research, synthesis of DNA and manufacture of DNA vectors. For example, the polynucleotides and polypeptides of the present invention may be employed aε reεearch reagentε and materials for discovery of treatments and diagnoεticε to human diεease.

This invention provides a method for identification of the receptor for hChat. The gene encoding the receptor can be identified by numerous methods known to those of skill in the art, for example, ligand panning and FACS sorting (Coligan, et al., Current Protocols in Immun. , 1(2), Chapter 5, (1991)). Preferably, expression cloning is employed wherein polyadenylated RNA is prepared from a cell responsive to hChat, and a cDNA library created from this RNA is divided into pools and used to transfect COS cells or other cells that are not responsive to hChat. Transfected cells which are grown on glass slides are exposed to labeled hChat. hChat can be labeled by a variety of means including iodination or inclusion of a recognition site for a site- specific protein kinase. Following fixation and incubation, the slides are subjected to autoradiographic analysis. Positive pools are identified and sub-pools are prepared and retransfected using an iterative sub-pooling and rescreening process, eventually yielding a single clone that encodes the putative receptor.

As an alternative approach for receptor identification, labeled hChat can be photoaffinity linked with cell membrane or extract preparations that express the receptor molecule. Cross-linked material is resolved by SDS-PAGE and exposed to X-ray film. The labeled complex containing the hChat- receptor can be excised, resolved into peptide fragments, and subjected to protein microεequencing. The amino acid εequence obtained from microsequencing would be used to design a set of degenerate oligonucleotide probes to screen a cDNA library to identify the gene encoding the putative receptor.

This invention also provides a method of screening compounds to identify those which specifically interact with, and enhance or block, the synthesiε of acetylcholine by choline acetyltransferase. An example of such a method includes incubating acetyl-CoA, choline, hChat and the potential agoniεt or antagonist under appropriate conditions such that hChat would normally catalyzes the transfer of the acyl-group to choline to yield acetylcholine. Results can be measured as pmol of acetylcholine formed per minute and per mg of protein. Control reactions could be performed in the absence of the agonist or antagonist such that an increased or decreased production of acetylcholine necesεitates the finding of the agonist or antagonist as effective.

A potential hChaT antagonist is an antibody, or in some cases, an oligonucleotide, which binds to hChat and blocks its interaction with acetyl-CoA. Potential antagonists may also be proteins which are closely related to hChat but impart no biological functions such that the synthesis of acetylcholine is prevented, e.g., fragments of hChat.

Potential hChaT antagonists also include antisense constructs prepared using antisenεe technology. Antisense technology can be used to control gene expression through triple-helix formation or antisense DNA or RNA, both of which methods are based on binding of a polynucleotide to DNA or RNA. For example, the 5' coding portion of the polynucleotide sequence, which encodes for the mature polypeptides of the present invention, is used to design an antisense RNA oligonucleotide of from about 10 to 40 base pairs in length. A DNA oligonucleotide is designed to be complementary to a region of the gene involved in transcription (triple helix -see Lee et al., Nucl. Acids Res., 6:3073 (1979); Cooney et al, Science, 241:456 (1988); and Dervan et al., Science, 251: 1360 (1991)), thereby preventing transcription and the production of hChat. The antisense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks translation of the mRNA molecule into the hChat polypeptide (antisenεe - Okano, J. Neurochem. , 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988)). The oligonucleotides described above can also be delivered to cells such that the antisense RNA or DNA may be expressed in vivo to inhibit production of hChat.

A potential antagonist includes a small molecule which binds to and occupies the catalytic site of the hChat polypeptide thereby making the catalytic site inaccessible to acetyl-CoA such that synthesis of acetylcholine is prevented. Exampleε of εmall moleculeε include, but are not limited to, small peptides or peptide-like moleculeε.

The antagonistε may be employed to treat Parkinson's Diseaεe, which produces a relative excess of acetylcholine because of the loss of dopamine. Similarly, accidental overdoses with various pharmaceuticals and contamination by toxins can also produce relative over-activity of the cholinergic system and may be treated with the antagonists of the present invention.

The antagonists may be employed in a composition with a pharmaceutically acceptable carrier, e.g., as hereinafter described.

The hChat polypeptides and agonists and antagonists may be employed in combination with a suitable pharmaceutical carrier. Such compositionε comprise a therapeutically effective amount of the polypeptide, and a pharmaceutically acceptable carrier or excipient. Such a carrier includes but is not limited to saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. The formulation should suit the mode of administration.

The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositionε of the invention. Aεεociated with εuch container(ε) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration. In addition, the pharmaceutical compositions may be employed in conjunction with other therapeutic compoundε.

These pharmaceutical compositions are administered in a convenient manner such as by the intravenous, intracerebral, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal routes. The pharmaceutical compositionε are administered in an amount which is effective for treating and/or prophylaxis of the specific indication. In general, the pharmaceutical compoεitionε will be adminiεtered in an amount of at leaεt about 10 μg/kg body weight and in moεt cases they will be adminiεtered in an amount not in exceεε of about 8 mg/Kg body weight per day. In moεt cases, the dosage is from about 10 μg/kg to about 1 mg/kg body weight daily, taking into account the routes of administration, symptoms, etc.

The hChat polypeptides, agonists and antagonists which are polypeptides may also be employed in accordance with the present invention by expresεion of such polypeptideε in vivo, which iε often referred to aε "gene therapy."

Thus, for example, cells from a patient may be engineered with a polynucleotide (DNA or RNA) encoding a polypeptide ex vivo, with the engineered cells then being provided to a patient to be treated with the polypeptide. Such methods are well-known in the art. For example, cells may be engineered by procedures known in the art by use of a retroviral particle containing RNA encoding a polypeptide of the present invention.

Similarly, cells may be engineered in vivo for expresεion of a polypeptide in vivo by, for example, procedures known in the art. As known in the art, a producer cell for producing a retroviral particle containing RNA encoding the polypeptide of the present invention may be administered to a patient for engineering cells in vivo and expresεion of the polypeptide in vivo. Theεe and other methodε for adminiεtering a polypeptide of the preεent invention by εuch method εhould be apparent to thoεe skilled in the art from the teachingε of the preεent invention. For example, the expression vehicle for engineering cells may be other than a retrovirus, for example, an adenovirus which may be used to engineer cells in vivo after combination with a suitable delivery vehicle.

Thiε invention iε also related to the use of the hChaT gene as part of a diagnostic assay for detecting diseaseε or susceptibility to diseases related to the presence of mutated hChaT. Such diseases are related to an acetylcholine deficiency, for example, neurological disorders. Individuals carrying mutations in the hChaT gene may be detected at the DNA level by a variety of techniques. Nucleic acids for diagnosis may be obtained from a patient's cells, such as from blood, tiεεue biopsy and autopsy material. The genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR (Saiki et al . , Nature, 324:163-166 (1986)) prior to analysiε. RNA or cDNA may also be used for the εame purpose. As an example, PCR primers complementary to the nucleic acid encoding hChaT can be used to identify and analyze hChaT mutations. For example, deletions and insertions can be detected by a change in size of the amplified product in comparison to the normal genotype. Point mutations can be identified by hybridizing amplified DNA to radiolabeled hChaT RNA or alternatively, radiolabeled hChaT antisenεe DNA εequences. Perfectly matched sequences can be distinguiεhed from miεmatched duplexeε by RNaεe A digeεtion or by differenceε in melting temperatures.

Genetic testing based on DNA sequence differenceε may be achieved by detection of alteration in electrophoretic mobility of DNA fragmentε in gelε with or without denaturing agents. Small sequence deletions and insertions can be visualized by high resolution gel electrophoresis. DNA fragments of different sequenceε may be diεtinguiεhed on denaturing formamide gradient gelε in which the mobilitieε of different DNA fragmentε are retarded in the gel at different poεitionε according to their εpecific melting or partial melting temperatures (see, e.g., Myers et al . , Science, 230:1242 (1985) ) .

Sequence changes at specific locations may also be revealed by nuclease protection aεεayε, εuch aε RNaεe and Si protection or the chemical cleavage method (e.g., Cotton et al . , PNAS, USA, 85:4397-4401 (1985)).

Thus, the detection of a specific DNA εequence may be achieved by methods such as hybridization, RNaεe protection, chemical cleavage, direct DNA εequencing or the use of restriction enzymes, (e.g., Restriction Fragment Length Polymorphismε (RFLP) ) and Southern blotting of genomic DNA. In addition to more conventional gel-electrophoreεis and DNA sequencing, mutations can also be detected by in si tu analysiε.

The preεent invention alεo relateε to a diagnostic assay for detecting altered levels of hChat protein in various tissues since an over-expression of the proteins compared to normal control tissue samples may detect the presence of a neurological disease or susceptibility to a neurological disease. Assays used to detect levels of hChat protein in a sample derived from a hoεt are well-known to thoεe of εkill in the art and include radioimmunoassays, competitive-binding assays, Western Blot analysis, ELISA assayε and "εandwich" assay. An ELISA assay (Coligan, et al., Current Protocols in Immunology, 1(2), Chapter 6, (1991)) initially comprises preparing an antibody specific to the hChat antigen, preferably a monoclonal antibody. In addition a reporter antibody is prepared against the monoclonal antibody. To the reporter antibody is attached a detectable reagent such as radioactivity, fluorescence or, in this example, a horεeradish peroxidase enzyme. A sample is removed from a host and incubated on a solid support, e.g. a polystyrene dish, that binds the proteins in the sample. Any free protein binding sites on the dish are then covered by incubating with a non-specific protein like BSA. Next, the monoclonal antibody is incubated in the dish during which time the monoclonal antibodies attach to any hChat proteins attached to the polystyrene dish. All unbound monoclonal antibody is washed out with buffer. The reporter antibody linked to horseradish peroxidase is now placed in the dish resulting in binding of the reporter antibody to any monoclonal antibody bound to hChat. Unattached reporter antibody is then washed out. Peroxidase substrates are then added to the dish and the amount of color developed in a given time period is a measurement of the amount of hChat protein present in a given volume of patient sample when compared against a standard curve.

A competition asεay may be employed wherein antibodieε specific to hChat are attached to a solid support and labeled hChat and a sample derived from the host are passed over the solid εupport and the amount of label detected, for example by liquid εcintillation chromatography, can be correlated to a quantity of hChat in the sample.

A "sandwich" aεεay iε similar to an ELISA assay. In a "sandwich" assay hChat is passed over a solid support and binds to antibody attached to a solid support. A second antibody is then bound to the hChat. A third antibody which iε labeled and εpecific to the εecond antibody iε then paεεed over the εolid εupport and bindε to the εecond antibody and an amount can then be quantitated.

The sequences of the present invention are also valuable for chromosome identification. The sequence is specifically targeted to and can hybridize with a particular location on an individual human chromosome. Moreover, there iε a current need for identifying particular sites on the chromosome. Few chromosome marking reagents based on actual sequence data (repeat polymorphisms) are presently available for marking chromosomal location. The mapping of DNAε to chromoεomes according to the present invention is an important first step in correlating those sequences with genes asεociated with disease.

Briefly, sequences can be mapped to chromosomeε by preparing PCR primerε (preferably 15-25 bp) from the cDNA. Computer analysis of the 3' untranslated region is used to rapidly select primers that do not span more than one exon in the genomic DNA, thus complicating the amplification process. These primers are then used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to the primer will yield an amplified fragment. PCR mapping of somatic cell hybrids is a rapid procedure for assigning a particular DNA to a particular chromosome. Using the present invention with the same oligonucleotide primers, sublocalization can be achieved with panels of fragments from specific chromosomes or pools of large genomic clones in an analogous manner. Other mapping strategies that can similarly be used to map to its chromosome include in si tu hybridization, prescreening with labeled flow-sorted chromosomes and preselection by hybridization to construct chromosome specific-cDNA libraries.

Fluorescence in si tu hybridization (FISH) of a cDNA clone to a metaphase chromoεomal spread can be uεed to provide a precise chromosomal location in one step. This technique can be used with cDNA as short as 500 or 600 bases,- however, clones larger than 2,000 bp have a higher likelihood of binding to a unique chromosomal location with sufficient signal intensity for simple detection. FISH requires use of the clones from which the EST was derived, and the longer the better. For example, 2,000 bp iε good, 4,000 iε better, and more than 4,000 iε probably not necessary to get good resultε a reaεonable percentage of the time. For a review of thiε technique, see Verma et al. , Human Chromosomes: a Manual of Basic Techniques, Pergamon Press, New York (1988) .

Once a sequence has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be correlated with genetic map data. Such data are found, for example, in V. McKusick, Mendelian Inheritance in Man (available on line through Johns Hopkins University Welch Medical Library) . The relationship between genes and diseases that have been mapped to the same chromosomal region are then identified through linkage analysis (coinheritance of physically adjacent genes) .

Next, it is necessary to determine the differences in the cDNA or genomic sequence between affected and unaffected individuals. If a mutation is observed in some or all of the affected individualε but not in any normal individualε, then the mutation iε likely to be the cauεative agent of the diseaεe.

With current reεolution of phyεical mapping and genetic mapping techniqueε, a cDNA precisely localized to a chromosomal region associated with the disease could be one of between 50 and 500 potential causative genes. (This asεumeε 1 megabaεe mapping resolution and one gene per 20 kb) .

The polypeptides, their fragments or other derivatives, or analogε thereof, or cellε expreεεing them can be uεed aε an immunogen to produce antibodies thereto. Theεe antibodies can be, for example, polyclonal or monoclonal antibodies. The present invention also includes chimeric, single chain, and humanized antibodies, as well as Fab fragments, or the product of an Fab expression library. Various procedures known in the art may be used for the production of such antibodies and fragmentε.

Antibodies generated against the polypeptides correεponding to a sequence of the present invention can be obtained by direct injection of the polypeptides into an animal or by administering the polypeptides to an animal, preferably a nonhuman. The antibody so obtained will then bind the polypeptides itself. In this manner, even a sequence encoding only a fragment of the polypeptideε can be uεed to generate antibodieε binding the whole native polypeptideε. Such antibodies can then be used to isolate the polypeptide from tisεue expreεεing that polypeptide.

For preparation of monoclonal antibodieε, any technique which provideε antibodies produced by continuous cell line cultures can be used. Examples include the hybridoma technique (Kohler and Milstein, 1975, Nature, 256:495-497), the trioma technique, the human B-cell hybridoma technique (Kozbor et al., 1983, Immunology Today 4:72), and the EBV- hybridoma technique to produce human monoclonal antibodieε (Cole, et al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).

Techniques described for the production of single chain antibodieε (U.S. Patent 4,946,778) can be adapted to produce single chain antibodies to immunogenic polypeptide products of this invention. Also, transgenic mice may be used to express humanized antibodies to immunogenic polypeptide products of thiε invention.

The preεent invention will be further deεcribed with reference to the following exampleε; however, it is to be understood that the present invention is not limited to such examples. All parts or amounts, unlesε otherwise specified, are by weight.

In order to facilitate understanding of the following examples certain frequently occurring methods and/or terms will be described.

"Plasmids" are designated by a lower case p preceded and/or followed by capital letters and/or numbers. The starting plasmids herein are either commercially available, publicly available on an unrestricted basis, or can be constructed from available plasmids in accord with published procedures. In addition, equivalent plasmids to those described are known in the art and will be apparent to the ordinarily skilled artisan.

"Digestion" of DNA refers to catalytic cleavage of the DNA with a restriction enzyme that acts only at certain sequenceε in the DNA. The variouε reεtriction enzymeε used herein are commercially available and their reaction conditions, cofactors and other requirements are uεed as would be known to the ordinarily skilled artisan. For analytical purposeε, typically 1 μg of plaεmid or DNA fragment iε used with about 2 units of enzyme in about 20 μl of buffer solution. For the purpose of isolating DNA fragments for plasmid construction, typically 5 to 50 μg of DNA are digested with 20 to 250 units of enzyme in a larger volume. Appropriate buffers and substrate amounts for particular restriction enzymes are specified by the manufacturer. Incubation times of about 1 hour at 37^*C are ordinarily used, but may vary in accordance with the supplier'ε instructions. After digestion the reaction is electrophoresed directly on a polyacrylamide gel to isolate the desired fragment.

Size separation of the cleaved fragments is performed using 8 percent polyacrylamide gel described by Goeddel, D. et al . , Nucleic Acids Res., 8:4057 (1980).

"Oligonucleotides" refers to either a single stranded polydeoxynucleotide or two complementary polydeoxynucleotide strandε which may be chemically synthesized. Such synthetic oligonucleotides have no 5' phosphate and thus will not ligate to another oligonucleotide without adding a phosphate with an ATP in the presence of a kinase. A synthetic oligonucleotide will ligate to a fragment that has not been dephosphorylated.

"Ligation" refers to the procesε of forming phoεphodieεter bondε between two double εtranded nucleic acid fragmentε (Maniatiε, T., et al., Id., p. 146). Unleεε otherwiεe provided, ligation may be accompliεhed uεing known bufferε and conditionε with 10 units to T4 DNA ligase ("ligase") per 0.5 μg of approximately equimolar amounts of the DNA fragments to be ligated.

Unlesε otherwiεe εtated, tranεformation waε performed aε deεcribed in the method of Graham, F. and Van der Eb, A., Virology, 52:456-457 (1973).

Example 1 Bacterial Expression and Purification of hChat

The DNA sequence encoding hChat, ATCC # 75856, is initially amplified using PCR oligonucleotide primers corresponding to the 5' and sequences of the procesεed hChat protein (minuε the εignal peptide εequence) and the vector sequences 3' to the hChat gene. Additional nucleotides corresponding to hChat are added to the 5' and 3 ' sequences respectively. The 5' oligonucleotide primer has the sequence 5' CGCGAGATCCACCATGAAGGCTTCCAGCCGCTTC 3' contains a Ba HI restriction enzyme site (underlined) followed by 21 nucleotides of hChat coding sequence starting from the presumed terminal amino acid of the processed protein codon. The 3' sequence 5' CGCGTCTAGAAGGGTACAGATGGTGGCC 3' contains complementary sequenceε to an Xbal site (underlined) and is followed by 18 nucleotides of hChat noncoding sequence located 3' to the hChat DNA insert. The restriction enzyme sites correspond to the restriction enzyme sites on the bacterial expression vector pQE-9 (Qiagen, Inc. 9259 Eton Avenue, Chatsworth, CA, 91311) . pQE-9 encodeε antibiotic resistance (Amp^r) , a bacterial origin of replication (ori) , an IPTG-regulatable promoter operator (P/0) , a ribosome binding site (RBS) , a 6-His tag and restriction enzyme sites. pQE-9 is then digested with BamHI and Xbal . The amplified sequences are ligated into pQE-9 and are inserted in frame with the sequence encoding for the histidine tag and the RBS. The ligation mixture is then used to transform E. coli strain ml5/REP4 available from Qiagen by the procedure described in Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, Cold Spring Laboratory Preεε, (1989) . M15/rep4 containε multiple copieε of the plaεmid pREP4, which expreεεeε the lad repreεsor and also confers kanamycin resistance (Kan^r) . Transfortnants are identified by their ability to grow on LB plateε and ampicillin/kanamycin reεistant colonies are selected. Plasmid DNA is isolated and confirmed by restriction analysis. Clones containing the desired constructs are grown overnight (0/N) in liquid culture in LB media supplemented with both Amp (100 ug/ml) and Kan (25 ug/ml) . The O/N culture is used to inoculate a large culture at a ratio of 1:100 to 1:250. The cells are grown to an optical density 600 (O.D.⁶⁰⁰) of between 0.4 and 0.6. IPTG ("Isopropyl-B-D-thiogalacto pyranoside") is then added to a final concentration of l mM. IPTG induces by inactivating the lad repressor, clearing the P/0 leading to increased gene expression. Cells are grown an extra 3 to 4 hours. Cells are then harvested by centrifugation. The cell pellet is solubilized in the chaotropic agent 6 Molar Guanidine HCl. After clarification, εolubilized hChat iε purified from this solution by chromatography on a Nickel- Chelate column under conditions that allow for tight binding by proteins containing the 6-His tag (Hochuli, E. et al. , J. Chromatography 411:177-184 (1984)) . hChat is eluted from the column in 6 molar guanidine HCl pH 5.0 and for the purpose of renaturation adjusted to 3 molar guanidine HCl, lOOmM sodium phosphate, 10 mmolar glutathione (reduced) and 2 mmolar glutathione (oxidized) . After incubation in this εolution for 12 hourε the protein iε dialyzed to 10 mmolar εodium phoεphate.

Example 2 Cloning and expreεεion of hChat using the baculovirus expresεion εvstem

The DNA sequence encoding the full length hChat protein, ATCC # 75856, is amplified using PCR oligonucleotide primers corresponding to the 5' and 3' sequenceε of the gene:

The 5' primer has the sequence 5' CGCGGGATCCACCATGAAGGCTTCCAGCCGCTTC 3' and containε a Bam HI reεtriction enzyme site (in bold) followed by 3 non-specific nucleotideε and 21 nucleotideε encoding hChat which resembles an efficient signal for the initiation of tranεlation in eukaryotic cellε (J. Mol. Biol. 1987, 19£, 947-950, Kozak, M.) .

The 3' primer has the sequence 5' CGCGGGTACCAGGTACAGATGGTGGCC 3' and contains the cleavage site for the restriction endonuclease Asp7l8 (in bold) followed by 16 nucleotides complementary to the coding region of hChat. The amplified εequenceε are isolated from a 1% agarose gel using a commercially available kit ("Geneclean, " BIO 101 Inc., La Jolla, Ca.) . The fragment is then digested with the endonucleaseε Bam Hi and Aεp7l8 and then purified as described above. Thiε fragment iε deεignated F2.

The vector pA35 (modification of pVL94l vector, diεcussed below) is used for the expression of the hChat protein using the baculovirus expression system (for review see: Summers, M.D. and Smith, G.E. 1987, A manual of methods for baculovirus vectors and insect cell culture procedures, Texas Agricultural Experimental Station Bulletin No. 1555) . This expresεion vector containε the εtrong polyhedrin promoter of the Autographa californica nuclear polyhedroεis virus (AcMNPV) followed by the recognition εites for the restriction endonucleaεeε Bam HI and Aεp718. The polyadenylation site of the simian viruε (SV)40 iε uεed for efficient polyadenylation. For an eaεy εelection of recombinant viruses the beta-galactosidase gene from E.coli is inserted in the same orientation as the polyhedrin promoter followed by the polyadenylation signal of the polyhedrin gene. The polyhedrin sequenceε are flanked at both εideε by viral εequenceε for the cell-mediated homologous recombination of cotransfected wild-type viral DNA. Many other baculovirus vectors could be used in place of pRGl such as pRGl, pAc373, pVL941 and pAcIMl (Luckow, V.A. and Summers, M.D. , Virology, 170:31-39).

The plasmid is digested with the restriction enzymeε Bam Hi and Aεp718 and then dephoεphorylated uεing calf intestinal phosphatase by procedures known in the art. The DNA is then isolated from a 1% agarose gel as described in Example l. This vector DNA is deεignated V2.

Fragment F2 and the dephosphorylated plasmid V2 are ligated with T4 DNA ligase. E.coli HB101 cells are then transformed and bacteria identified that contained the plasmid (pBac hChat) with the hChat gene using the enzymes Bam HI and Asp718. The sequence of the cloned fragment is confirmed by DNA sequencing.

5 μg of the plasmid pBac hChat are cotransfected with 1.0 μg of a commercially available linearized baculovirus ("BaculoGold™ baculovirus DNA", Pharmingen, San Diego, CA.) using the lipofection method (Feigner et al. Proc. Natl. Acad. Sci. USA, 84:7413-7417 (1987)). lμg of BaculoGold™ virus DNA and 5 μg of the plasmid pBac hChat are mixed in a sterile well of a microtiter plate containing 50 μl of serum free Grace's medium (Life Technologies Inc., Gaithersburg, MD) . Afterwards 10 μl Lipofectin plus 90 μl Grace's medium are added, mixed and incubated for 15 minuteε at room temperature. Then the transfection mixture is added drop-wise to the Sf9 inεect cells (ATCC CRL 1711) seeded in a 35 mm tisεue culture plate with l ml Grace' medium without serum. The plate is rocked back and forth to mix the newly added solution. The plate is then incubated for 5 hours at 27°C. After 5 hours the transfection εolution is removed from the plate and 1 ml of Grace's insect medium supplemented with 10% fetal calf serum iε added. The plate iε put back into an incubator and cultivation continued at 27°C for four dayε.

After four days the supernatant iε collected and a plaque assay performed similar aε described by Summers and Smith (supra) . As a modification an agarose gel with "Blue Gal" (Life Technologies Inc., Gaithersburg) is used which allowε an easy isolation of blue εtained plaqueε. (A detailed deεcription of a "plaque aεsay" can also be found in the user's guide for insect cell culture and baculovirology distributed by Life Technologies Inc., Gaithersburg, page 9- 10) .

Four days after the serial dilution of the viruses is added to the cells, blue stained plaques are picked with the tip of an Eppendorf pipette. The agar containing the recombinant viruses is then resuεpended in an Eppendorf tube containing 200 μl of Grace's medium. The agar is removed by a brief centrifugation and the supernatant containing the recombinant baculoviruses iε uεed to infect Sf9 cells seeded in 35 mm dishe . Four days later the supernatantε of theεe culture disheε are harvested and then stored at 4°C.

Sf9 cellε are grown in Grace'ε medium εupplemented with 10% heat-inactivated FBS. The cells are infected with the recombinant baculovirus V-hChat at a multiplicity of infection (MOD of 2. Six hours later the medium is removed and replaced with SF900 II medium minus methionine and cysteine (Life Technologies Inc., Gaithersburg). 42 hours later 5 μCi of ³⁵S-methionine and 5 μCi ³⁵S cysteine (Amersham) are added. The cells are further incubated for 16 hours before they are harvested by centrifugation and the labelled proteins visualized by SDS-PAGE and autoradiography.

Example 3 Expreεεion of Recombinant hChat in COS cells

The expresεion of plasmid, hChat HA is derived from a vector pcDNAI/Amp (Invitrogen) containing: 1) SV40 origin of replication, 2) ampicillin resistance gene, 3) E.coli replication origin, 4) CMV promoter followed by a polylinker region, a SV40 intron and polyadenylation site. A DNA fragment encoding the entire hChat precursor and a HA tag fused in frame to its 3' end is cloned into the polylinker region of the vector, therefore, the recombinant protein expression is directed under the CMV promoter. The HA tag correspond to an epitope derived from the influenza hemagglutinin protein as previously described (I. Wilson, H. Niman, R. Heighten, A Cherenson, M. Connolly, and R. Lerner, 1984, Cell 37, 767) . The infusion of HA tag to our target protein allows easy detection of the recombinant protein with an antibody that recognizes the HA epitope.

The plasmid construction strategy is deεcribed aε followε:

The DNA εequence encoding hChat, ATCC# 75856, iε constructed by PCR on the original EST cloned using two primers: the 5' primer 5' CGαMGATCCACC-ATGAAGGCITCCAGCCGCTTC 3' contains a Bam HI site (bold) followed by 21 nucleotides of hChat coding sequence starting from the initiation codon; the 3' sequence 5' CGCGTCTAGATCAAGCGTAGTCTGGGACGTCGTATGGG TAGCCTCTGCACTCAGCCCC 3' containε complementary sequenceε to an Xba I site (bold) , translation stop codon, HA tag and the laεt 18 nucleotideε of the hChat coding εequence (not including the εtop codon) . Therefore, the PCR product contains a Bam Hi site, hChat coding sequence followed by HA tag fused in frame, a translation termination stop codon next to the HA tag, and an Xba I site. The PCR amplified DNA fragment and the vector, pcDNAI/Amp, are digested with Bam Hi and Xba I reεtriction enzyme and ligated. The ligation mixture is transformed into E. coli strain SURE (available from Stratagene Cloning Systems, 11099 North Torrey Pines Road, La Jolla, CA 92037) the transformed culture is plated on ampicillin media plates and resistant colonies are selected. Plasmid DNA is isolated from tranεformantε and examined by reεtriction analyεiε for the preεence of the correct fragment. For expression of the recombinant hChat, COS cells are transfected with the expresεion vector by DEAE- DEXTRAN method (J. Sambrook, E. Fritsch, T. Maniatiε, Molecular Cloning: A Laboratory Manual, Cold Spring Laboratory Preεε, (1989)). The expression of the hChat HA protein is detected by radiolabelling and immunoprecipitation method (E. Harlow, D. Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, (1988)). Cells are labelled for 8 hours with ³⁵S-cysteine two days post transfection. Culture media are then collected and cells are lysed with detergent (RIPA buffer (150 mM NaCl, 1% NP-40, 0.1% SDS, 1% NP-40, 0.5% DOC, 50mM Tris, pH 7.5) (Wilson, I. et al., Id. 37:767 (1984)). Both cell lysate and culture media are precipitated with a HA εpecific monoclonal antibody. Proteinε precipitated are analyzed on 15% SDS-PAGE gels.

Example 4 Expresεion pattern of hChat in human tissue

Northern blot analysis is carried out to examine the levels of expression of hChat in human tissueε. Total cellular RNA εamples are iεolated with RNAzol™ B εystem (Biotecx Laboratories. Inc. 6023 South Loop East, Houston, TX 77033) . About 10μg of total RNA isolated from each human tissue specified is separated on 1% agarose gel and blotted onto a nylon filter. (Sambrook, Fritsch, and Maniatis, Molecular Cloning, Cold Spring Harbor Preεε, (1989)). The labeling reaction iε done according to the Stratagene Prime- It kit with 50ng DNA fragment. The labeled DNA iε purified with a Select-G-50 column. (5 Prime - 3 Prime, Inc. 5603 Arapahoe Road, Boulder, CO 80303) . The filter iε then hybridized with radioactive labeled full length hChat gene at 1,000,000 cpm/ml in 0.5 M NaP0₄, pH 7.4 and 7% SDS overnight at 65^*C. After wash twice at room temperature and twice at 60'C with 0.5 x SSC, 0.1% SDS, the filter is then exposed at -70"C overnight with an intenεifying screen. The message RNA for hChat is abundant in liver, skeletal muscle, pancreas and to a lesser degree in heart, kidney and placenta (Figure 2) . Numerous modifications and variations of the present invention are posεible in light of the above teachingε and, therefore, within the εcope of the appended claims, the invention may be practiced otherwise than aε particularly described.

SEQUENCE LISTING

(1) GENERAL INFORMATION: (i) APPLICANT: HUDSON, ET AL.

(ii) TITLE OF INVENTION: Human Choline Acetyltransferase

(iii) NUMBER OF SEQUENCES: 2

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: CARELLA, BYRNE, BAIN, GILFILLAN,

CECCHI, STEWART & OLSTEIN

(B) STREET: 6 BECKER FARM ROAD

(C) CITY: ROSELAND

(D) STATE: NEW JERSEY

(E) COUNTRY: USA

(F) ZIP: 07068

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: 3.5 INCH DISKETTE

(B) COMPUTER: IBM PS/2

(C) OPERATING SYSTEM: MS-DOS

(D) SOFTWARE: WORD PERFECT 5.1

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE: Submitted herewith

(C) CLASSIFICATION:

(vii) PRIOR APPLICATION DATA

(A) APPLICATION NUMBER:

(B) FILING DATE: (viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: FERRARO, GREGORY D.

(B) REGISTRATION NUMBER: 36,134

(C) REFERENCE/DOCKET NUMBER: 325800-209

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 201-994-1700

(B) TELEFAX: 201-994-1744

(2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 2034 BASE PAIRS

(B) TYPE: NUCLEIC ACID

(C) STRANDEDNESS: SINGLE

(D) TOPOLOGY: LINEAR

(ϋ) MOLECULE TYPE: CDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

ATGAAGGCTT CCAGCCGCTT CAAGGCACAC CAGGATGCAC TGCCACGGT GCCCGTGCCC 60

CCTCTCCAGC AGTCCCTGGA CCACTACCTG AAGGCGCTGC AGCCCATCGT GAGTGAGGAG 120

GAGTGGGCCC ACACCAAGCA GCTGGTGGAT GAGTTTCAGG CCTCAGGAGG TGTAGGGGAG 180

CGCCTGCAGA AGGGGCTGGA GCGTCGGGCC AGGAAGACGG AGAACTGGCT GTCTGAGTGG 240

TGGCTCAAGA CCGCCTACCT CCAGTACCGC CAGCCTGTGG TCATCTACTC GAGCCCAGGC 300

GTGATGCTAC CCAAGCAGGA CTTCGTGGAC CTGCAGGGTC AGCTCCGATT TGCTGCCAAA 360

CTCATTGAGG GTGTGTTGGT TTTCAAGGTC ATGATTGACA ACGAGACCCT GCCCGTGGAG 420

TACCTGGGGG GGAAGCCACT GTGCATGAAC CAGTTCTATC AGATCTTGTC CTCCTGCCGA 480

GTGCCGGGCC CCAAGCAGGA CACAGTCAGC AACTTCAGCA AGACCAAGAA GCCTCCCACG 540

CACATCACCG TGGTACACAA CTACCAGTTT TTTGAGCTGG ATGTGTACCA CAGTGACGGG 600

ACACCCCTCA CTGCGGATCA GATCTTTGTG CAGCTGGAGA AGATCTGGAA CTCATCCCTA 660

CAGACCAACA AGGAGCCTGT GGGCATCCTC ACCTCCAACC ACCGCAACTC CTGGGCCAAG 720

GCATACAACA CCCTCATCAA AGACAAGGTG AACCGGGATT CCGTGCGCTC CATCCAGAAG 780

AGCATCTTCA CCGTGTGCCT AGATGCAACC ATGCCCAGGG TCTCAGAAGA CGTGTACCGC 840

AGCCACGTGG CAGGCCAGAT GCTGCATGGG GGCGGCAGCA GGCTCAACAG CGGCAACCGC 900

TGGTTCGACA AGACGCTGCA GTTCATCGTG GCAGAAGATG GCTCCTGTGG GCTTGTGTAC 960

GAGCATGCTG CAGCGGAGGG GCCCCCTATT GTCACCCTTC TGGACTATGT CATCGAGTAC 1020 ACGAAGAAAC CCGAGCTTGT GCGGTCTCCC ATGGTGCCCC TGCCCATGCC CAAGAAGCTG 1080 CGGTTCAACA TCACCCCCGA GATCAAGAGC GACATCGAGA AGGCCAAGCA GAACCTCAGC 1140 ATCATGATCC AGGACCTGGA TATCACCGTG ATGGTGTTCC ACCATTTTGG AAAAGACTTC 1200 CCCAAGTCGG AGAAGCTAAG CCCAGATGCC TTCATCCAGA TGGCTTTGCA GCTGGCCTAC 1260 TACAGGTTCT ACGGAAAGGA ATGTGCCACC TATGAAAGTG CCTCCCTGCG CATGTTTCAC 1320 CTGGGGCGCA CCGACACCAT CCGCTCGGGT TCCATGGACT CACTCACCTT TGTCAAGGCC 1380 ATGGATGACT CCAGCGTCAC GGAGCACCAG AAGGTGGAGC TGCTGCGGAA GGCCGTGCAG 1440 GCCCACCGAG GTTACACCGA CCGGGCCATC CGGGGGGAGG GCTTTGATCG ACACCTGCTG 1500 GGCCTGAAGC TGCAGGCCAT CGAGGACCTG GTGAGCATGC CCGACATCTT CATGGACACC 1560 TCCTACGCCA TCGGCATGTA CTTCCACCTC TCCACCAGGC AGGTCCCTGC CAAGAAGATG 1620 TGTCATGTTC TTCGGGCCCG TGGTCCCCGA CGGGTACGGT GTCTTTATAA CCCCATGGAG 1680 GGCCACATCA ACTTCTCCCT GTCGGACTAC AAAAGGTGGG GGGAGACCAA CGCCGCCCGC 1740 CTGGTGTATT ACCTGGAGAA GGCGCTCCTG GACATGCGTG CCCTGGGTGA GAGCCACCCC 1800 CGGGCCAAGT TCTTAGCCCC TAGGACTCAG GCCTGCCAAT GCCACAGGCA AGCCCACCCT 1860 AGGATGGGCC ACCCACCAGG GATCAGCTCC TTGGTTCCCT CTTCCTTGGT TCCCTCTTCC 1920 CTGGTCCCCC CAATTCTACT GAGCCACGGA CCGCATCCTC CAGGGGGCTG CAGGCCCAGC 1980 CAAGTGCCTT CCGTGGGTCA TCCCAGCACC TGCCAGGGCC CGACCTGGGG CTGA 2034

(2) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 677 AMINO ACIDS

(B) TYPE: AMINO ACID

(C) STRANDEDNESS:

(D) TOPOLOGY: LINEAR

(ii) MOLECULE TYPE: PROTEIN

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

Met Lys Ala Ser Ser Arg Phe Lys Ala His Gin Asp Ala Leu Pro

5 10 15

Arg Leu Pro Val Pro Pro Leu Gin Gin Ser Leu Asp His Tyr Lys

20 25 30

Leu Lys Ala Leu Gin Pro lie Val Ser Glu Glu Glu Trp Ala His

35 40 45

Thr Lys Gin Leu Val Asp Glu Phe Gin Ala Ser Gly Gly Val Gly

50 55 60 Glu Arg Leu Gin Lys Gly Leu Glu Arg Arg Ala Arg Lys Thr Glu

65 70 75

Asn Trp Leu Ser Glu Trp Trp Leu Lys Thr Ala Tyr Leu Gin Tyr

80 85 90

Arg Gin Pro Val Val lie Tyr Ser Ser Pro Gly Val Met Leu Pro

95 100 105

Lys Gin Aεp Phe Val Aεp Leu Gin Gly Gin Leu Arg Phe Ala Ala

110 115 120

Lyε Leu lie Glu Gly Val Leu Val Phe Lyε Val Met lie Asp Asn

125 130 135

Glu Thr Leu Pro Val Glu Tyr Leu Gly Gly Lyε Pro Leu Cys Met

140 145 150

Asn Gin Phe Tyr Gin lie Leu Ser Ser Cys Arg Val Pro Gly Pro

155 160 165

Lys Gin Asp Thr Val Ser Asn Phe Ser Lys Thr Lys Lyε Pro Pro

170 175 180

Thr His lie Thr Val Val His Asn Tyr Gin Phe Phe Glu Leu Asp

185 190 195

Val Tyr His Ser Asp Gly Thr Pro Leu Thr Ala Asp Gin lie Phe

200 205 210

Val Gin Leu Glu Lys lie Trp Asn Ser Ser Leu Gin Thr Asn Lys

215 220 225

Glu Pro Val Gly lie Leu Thr Ser Asn His Arg Asn Ser Trp Ala

230 235 240

Lys Ala Tyr Asn Thr Leu lie Lys Asp Lyε Val Asn Arg Asp Ser

245 250 255

Val Arg Ser lie Gin Lys Ser lie Phe Thr Val Cys Leu Asp Ala

260 265 270

Thr Met Pro Arg Val Ser Glu Asp Val Tyr Arg Ser His Val Ala

275 280 285

Gly Gin Met Leu His Gly Gly Gly Ser Arg Leu Asn Ser Gly Asn

290 295 300

Arg Trp Phe Asp Lys Thr Leu Gin Phe lie Val Ala Glu Asp Gly

305 310 315 Ser Cys Gly Leu Val Tyr Glu Hiε Ala Ala Ala Glu Gly Pro Pro

320 325 330 lie Val Thr Leu Leu Asp Tyr Val lie Glu Tyr Thr Lys Lys Pro

335 340 345

Glu Leu Val Arg Ser Pro Met Val Pro Leu Pro Met Pro Lys Lys

350 355 360

Leu Arg Phe Asn lie Thr Pro Glu lie Lys Ser Asp lie Glu Lys

365 370 375

Ala Lys Gin Asn Leu Ser lie Met lie Gin Asp Leu Asp lie Thr

380 385 390

Val Met Val Phe His His Phe Gly Lys Asp Phe Pro Lys Ser Glu

395 400 405

Lys Leu Ser Pro Aεp Ala Phe lie Gin Met Ala Leu Gin Leu Ala

410 415 420

Tyr Tyr Arg Phe Tyr Gly Lys Glu Cys Ala Thr Tyr Glu Ser Ala

425 430 435

Ser Leu Arg Met Phe His Leu Gly Arg Thr Asp Thr lie Arg Ser

440 445 450

Gly Ser Met Asp Ser Leu Thr Phe Val Lys Ala Met Asp Asp Ser

455 460 465

Ser Val Thr Glu Hiε Gin Lys Val Glu Leu Leu Arg Lys Ala Val

470 475 480

Gin Ala His Arg Gly Tyr Thr Asp Arg Ala lie Arg Gly Glu Gly

485 490 495

Phe Asp Arg His Leu Leu Gly Leu Lys Leu Gin Ala lie Glu Asp

500 505 510

Leu Val Ser Met Pro Asp lie Phe Met Aεp Thr Ser Tyr Ala lie

515 520 525

Gly Met Tyr Phe Hiε Leu Ser Thr Arg Gin Val Pro Ala Lyε Lyε

530 535 540

Met Cyε His Val Leu Arg Ala Arg Gly Pro Arg Arg Val Cys Leu

545 550 555

Tyr Asn Pro Met Glu Gly His lie Asn Phe Ser Leu Ser Asp Tyr

560 565 570 Lys Arg Trp Gly Glu Thr Asn Ala Ala Arg Leu Val Tyr Tyr Leu

575 580 585

Gly Lys Ala Leu Leu Asp Met Arg Ala Leu Val Lys Ser His Pro

590 595 600

Arg Ala Lys Phe Leu Ala Pro Arg Thr Gin Ala Cyε Gin Cys His

605 610 615

Arg Gin Ala His Pro Arg Met Gly His Pro Pro Gly lie Ser Ser

620 625 630

Leu Val Pro Ser Ser Leu Val Pro Ser Ser Leu Val Pro Pro lie

635 640 645

Leu Leu Ser His Gly Pro His Pro Pro Gly Gly Cys Arg Pro Ser

650 655 660

Gin Val Pro Ser Val Gly His Pro Ser Thr Cyε Gin Gly Pro Thr

665 670 675 Trp Gly

Claims

WHAT IS CLAIMED IS:

1. An isolated polynucleotide selected from the group consisting of :

(a) a polynucleotide encoding the polypeptide having the deduced amino acid sequence of Figure 1 or a fragment, analog or derivative of said polypeptide;

(b) a polynucleotide encoding the polypeptide having the amino acid sequence encoded by the cDNA contained in ATCC Deposit No. 75856 or a fragment, analog or derivative of said polypeptide.

2. The polynucleotide of Claim 1 wherein the polynucleotide is DNA.

3. The polynucleotide of Claim 1 wherein the polynucleotide iε RNA.

4. The polynucleotide of Claim 1 wherein the polynucleotide is genomic DNA.

5. The polynucleotide of Claim 2 wherein said polynucleotide encodes a polypeptide having the deduced amino acid sequence of Figure 1.

6. The polynucleotide of Claim 2 wherein said polynucleotide encodes the polypeptide encoded by the cDNA of ATCC Deposit No. 75856.

7. The polynucleotide of Claim 1 having the coding sequence shown in Figure 1.

8. The polynucleotide of Claim 2 having the coding sequence of ATCC Deposit No. 75856.

9. A vector containing the DNA of Claim 2.

10.A host cell genetically engineered with the vector of Claim 9.

11. A procesε for producing a polypeptide compriεing: expressing from the host cell of Claim 10 the polypeptide encoded by said DNA.

12. A process for producing cells capable of expressing a polypeptide comprising genetically engineering cells with the vector of Claim 9.

13. An isolated DNA hybridizable to the DNA of Claim 2 and encoding a polypeptide having hChat activity.

14. A polypeptide selected from the group consiεting of (i) a polypeptide having the deduced amino acid εequence of Figure 1 and fragmentε, analogε and derivativeε thereof and (ii) a polypeptide encoded by the cDNA of ATCC Depoεit No. 75856 and fragments, analogs and derivatives of said polypeptide.

15. The polypeptide of Claim 14 wherein the

- polypeptide has the deduced amino acid sequence of Figure 1.

16. An antibody against the polypeptide of claim 14.

17. An antagonist against the polypeptide of claim 14.

18. A method for the treatment of a patient having need of hChat comprising: administering to the patient a therapeutically effective amount of the polypeptide of claim 14.

19. A pharmaceutical composition compriεing the polypeptide of Claim 14 and a pharmaceutically acceptable carrier.

20. The method of Claim 18 wherein said therapeutically effective amount of the polypeptide is administered by providing to the patient DNA encoding εaid polypeptide and expreεεing εaid polypeptide in vivo.

21. A proceεε for identifying antagoniεtε and agoniεtε compriεing: combining hCHAT acetyl-CoA, choline and a compound to be εcreened under conditions where hChat catalyzes the transfer of the acyl-group to choline to yield acetylcholine; meaεuring the concentration of acetylcholine εyntheεized; and determining whether the compound enhanceε or blocks the production of acetylcholine.

22. A process for diagnosing a disease or susceptibility to diseaεe related to a mutation in the ChaT nucleic acid εequence compriεing: isolating a nucleic acid sequence encoding a ChaT from a εample derived from a host; and determining a mutation in the ChaT nucleic acid εequence.

23. A diagnoεtic proceεs comprising: analyzing for the presence of the polypeptide of claim 14 in a sample derived from a host.