Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/10—Transferases (2.)
  • C12N9/1025—Acyltransferases (2.3)
  • C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/02—Drugs for disorders of the nervous system for peripheral neuropathies
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• 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.
• 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.
• a novel mature polypeptide which is hChaT, as well as biologically active and diagnostically or therapeutically useful fragments, analogs and derivatives thereof.
• 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.
• 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.
• 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.
• ALS amyotrophic lateral sclerosis
• Alzheimer's Disease familial disautonomia
• Huntington's Disease Huntington's Disease
• mental retardation memory loss
• myasthenia gravis myasthenia gravis
• nucleic acid probes comprising nucleic acid molecules of sufficient length to specifically hybridize to hChaT sequences.
• 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.
• 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.
• 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.
• nucleic acid 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.
• 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.
• 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.
• 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.
• 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- hi ⁇ tidine 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 .
• 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.
• fragment 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.
• 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) .
• a conserved or non-conserved amino acid residue preferably a conserved amino acid residue
• substituted amino acid residue may or may not be one encoded by the genetic code
• one or more of the amino acid residues includes a substituent group
• the mature polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (
• polypeptides and polynucleotides of the present invention are preferably provided in an isolated form, and preferably are purified to homogeneity.
• isolated means that the material is removed from its original environment (e.g., the natural environment if it is naturally occurring) .
• 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.
• 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.
• 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.
• 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.
• promoter for example, 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.
• 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.
• 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 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.
• the construct further comprises regulatory sequences, including, for example, a promoter, operably linked to the sequence.
• a promoter operably linked to the sequence.
• 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) .
• 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.
• 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) ) .
• constructs in host cells can be used in a conventional manner to produce the gene product encoded by the recombinant sequence.
• 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.
• 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.
• 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.
• 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.
• PGK 3-phosphoglycerate kinase
• the heterologous structural sequence is assembled in appropriate phase with translation initiation and termination sequences.
• 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 Pseudomona ⁇ , Streptomyce ⁇ , and Staphylococcus, although others may also be employed as a matter of choice.
• 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) .
• 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) .
• pBR322 "backbone" sections are combined with an appropriate promoter and the structural sequence to be expressed.
• 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.
• mammalian cell culture systems can also be employed to express recombinant protein.
• 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, tran ⁇ criptional termination sequences, and 5' flanking nontranscribed sequence ⁇ . 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 culture ⁇ 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.
• HPLC high performance liquid chromatography
• 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 ho ⁇ t (for example, by bacterial, yeast, higher plant, insect and mammalian cells in culture) .
• a prokaryotic or eukaryotic ho ⁇ t for example, by bacterial, yeast, higher plant, insect and mammalian cells in culture
• 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 polypeptide ⁇ of the pre ⁇ ent 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.
• the hChat polypeptide ⁇ of the pre ⁇ ent invention may be employed to treat amyotrophic lateral sclerosis, Alzheimer's Disea ⁇ e, 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.
• Example ⁇ of ⁇ uch di ⁇ order ⁇ include gut and GI di ⁇ order ⁇ , cord disorders, including movement, continence and sen ⁇ ation, stem disorders, including sleep, blood pres ⁇ ure, respiration, and balance, hypothalamus disorders, including temperature, respiration, and endocrine function, and limbic sy ⁇ tem 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.
• polypeptides or polynucleotides encoding such polypeptides, for in vitro purposes related to scientific research, synthesis of DNA and manufacture of DNA vectors.
• the polynucleotides and polypeptides of the present invention may be employed a ⁇ re ⁇ earch reagent ⁇ and materials for discovery of treatments and diagno ⁇ tic ⁇ to human di ⁇ ease.
• 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)).
• 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.
• 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 micro ⁇ equencing. The amino acid ⁇ equence 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 synthesi ⁇ of acetylcholine by choline acetyltransferase.
• An example of such a method includes incubating acetyl-CoA, choline, hChat and the potential agoni ⁇ t 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 neces ⁇ itates 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 antisen ⁇ e 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.
• 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.
• the antisense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks translation of the mRNA molecule into the hChat polypeptide (antisen ⁇ e - 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.
• Example ⁇ of ⁇ mall molecule ⁇ include, but are not limited to, small peptides or peptide-like molecule ⁇ .
• the antagonist ⁇ may be employed to treat Parkinson's Disea ⁇ e, 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.
• a suitable pharmaceutical carrier includes but is not limited to saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof.
• 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 composition ⁇ of the invention.
• a ⁇ ociated with ⁇ uch container( ⁇ ) 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.
• the pharmaceutical compositions may be employed in conjunction with other therapeutic compound ⁇ .
• compositions are administered in a convenient manner such as by the intravenous, intracerebral, intraperitoneal, intramuscular, subcutaneous, intranasal or intradermal routes.
• the pharmaceutical composition ⁇ are administered in an amount which is effective for treating and/or prophylaxis of the specific indication.
• the pharmaceutical compo ⁇ ition ⁇ will be admini ⁇ tered in an amount of at lea ⁇ t about 10 ⁇ g/kg body weight and in mo ⁇ t cases they will be admini ⁇ tered in an amount not in exce ⁇ of about 8 mg/Kg body weight per day.
• the dosage is from about 10 ⁇ g/kg to about 1 mg/kg body weight daily, taking into account the routes of administration, symptoms, etc.
• hChat polypeptides, agonists and antagonists which are polypeptides may also be employed in accordance with the present invention by expres ⁇ ion of such polypeptide ⁇ in vivo, which i ⁇ often referred to a ⁇ "gene therapy.”
• 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.
• a polynucleotide DNA or RNA
• 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.
• cells may be engineered in vivo for expres ⁇ ion of a polypeptide in vivo by, for example, procedures 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 expres ⁇ ion of the polypeptide in vivo.
• the ⁇ e and other method ⁇ for admini ⁇ tering a polypeptide of the pre ⁇ ent invention by ⁇ uch method ⁇ hould be apparent to tho ⁇ e skilled in the art from the teaching ⁇ of the pre ⁇ ent invention.
• 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.
• Thi ⁇ invention i ⁇ also related to the use of the hChaT gene as part of a diagnostic assay for detecting disease ⁇ 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, ti ⁇ ue 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 analysi ⁇ .
• RNA or cDNA may also be used for the ⁇ ame purpose.
• 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 antisen ⁇ e DNA ⁇ equences. Perfectly matched sequences can be distingui ⁇ hed from mi ⁇ matched duplexe ⁇ by RNa ⁇ e A dige ⁇ tion or by difference ⁇ in melting temperatures.
• DNA sequence difference ⁇ may be achieved by detection of alteration in electrophoretic mobility of DNA fragment ⁇ in gel ⁇ with or without denaturing agents. Small sequence deletions and insertions can be visualized by high resolution gel electrophoresis. DNA fragments of different sequence ⁇ may be di ⁇ tingui ⁇ hed on denaturing formamide gradient gel ⁇ in which the mobilitie ⁇ of different DNA fragment ⁇ are retarded in the gel at different po ⁇ ition ⁇ according to their ⁇ pecific 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 a ⁇ ay ⁇ , ⁇ uch a ⁇ RNa ⁇ e and Si protection or the chemical cleavage method (e.g., Cotton et al . , PNAS, USA, 85:4397-4401 (1985)).
• the detection of a specific DNA ⁇ equence may be achieved by methods such as hybridization, RNa ⁇ e protection, chemical cleavage, direct DNA ⁇ equencing or the use of restriction enzymes, (e.g., Restriction Fragment Length Polymorphism ⁇ (RFLP) ) and Southern blotting of genomic DNA.
• restriction enzymes e.g., Restriction Fragment Length Polymorphism ⁇ (RFLP)
• RFLP Restriction Fragment Length Polymorphism ⁇
• mutations can also be detected by in si tu analysi ⁇ .
• the pre ⁇ ent invention al ⁇ o relate ⁇ 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 ho ⁇ t are well-known to tho ⁇ e of ⁇ kill in the art and include radioimmunoassays, competitive-binding assays, Western Blot analysis, ELISA assay ⁇ and " ⁇ andwich" 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.
• a reporter antibody is prepared against the monoclonal antibody.
• a detectable reagent such as radioactivity, fluorescence or, in this example, a hor ⁇ eradish 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.
• 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 as ⁇ ay may be employed wherein antibodie ⁇ specific to hChat are attached to a solid support and labeled hChat and a sample derived from the host are passed over the solid ⁇ upport and the amount of label detected, for example by liquid ⁇ cintillation chromatography, can be correlated to a quantity of hChat in the sample.
• 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 i ⁇ labeled and ⁇ pecific to the ⁇ econd antibody i ⁇ then pa ⁇ ed over the ⁇ olid ⁇ upport and bind ⁇ to the ⁇ econd antibody and an amount can then be quantitated.
• 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.
• Few chromosome marking reagents based on actual sequence data (repeat polymorphisms) are presently available for marking chromosomal location.
• the mapping of DNA ⁇ to chromo ⁇ omes according to the present invention is an important first step in correlating those sequences with genes as ⁇ ociated with disease.
• sequences can be mapped to chromosome ⁇ by preparing PCR primer ⁇ (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.
• 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 chromo ⁇ omal spread can be u ⁇ ed 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 i ⁇ good, 4,000 i ⁇ better, and more than 4,000 i ⁇ probably not necessary to get good result ⁇ a rea ⁇ onable percentage of the time.
• thi ⁇ technique see Verma et al. , Human Chromosomes: a Manual of Basic Techniques, Pergamon Press, New York (1988) .
• a cDNA precisely localized to a chromosomal region associated with the disease could be one of between 50 and 500 potential causative genes. (This as ⁇ ume ⁇ 1 megaba ⁇ e mapping resolution and one gene per 20 kb) .
• the polypeptides, their fragments or other derivatives, or analog ⁇ thereof, or cell ⁇ expre ⁇ ing them can be u ⁇ ed a ⁇ an immunogen to produce antibodies thereto.
• the ⁇ e 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 fragment ⁇ .
• Antibodies generated against the polypeptides corre ⁇ ponding 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 polypeptide ⁇ can be u ⁇ ed to generate antibodie ⁇ binding the whole native polypeptide ⁇ . Such antibodies can then be used to isolate the polypeptide from tis ⁇ ue expre ⁇ ing that polypeptide.
• any technique which provide ⁇ 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 antibodie ⁇ (Cole, et al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).
• 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.
• 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 sequence ⁇ in the DNA.
• the variou ⁇ re ⁇ triction enzyme ⁇ used herein are commercially available and their reaction conditions, cofactors and other requirements are u ⁇ ed as would be known to the ordinarily skilled artisan.
• For analytical purpose ⁇ typically 1 ⁇ g of pla ⁇ mid or DNA fragment i ⁇ used with about 2 units of enzyme in about 20 ⁇ l of buffer solution.
• 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 strand ⁇ 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.
• Ligase refers to the proces ⁇ of forming pho ⁇ phodie ⁇ ter bond ⁇ between two double ⁇ tranded nucleic acid fragment ⁇ (Maniati ⁇ , T., et al., Id., p. 146). Unle ⁇ otherwi ⁇ e provided, ligation may be accompli ⁇ hed u ⁇ ing known buffer ⁇ and condition ⁇ with 10 units to T4 DNA ligase ("ligase”) per 0.5 ⁇ g of approximately equimolar amounts of the DNA fragments to be ligated.
• ligase T4 DNA ligase
• the DNA sequence encoding hChat is initially amplified using PCR oligonucleotide primers corresponding to the 5' and sequences of the proces ⁇ ed hChat protein (minu ⁇ the ⁇ ignal peptide ⁇ equence) 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 sequence ⁇ 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.
• pQE-9 encode ⁇ 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.
• M15/rep4 contain ⁇ multiple copie ⁇ of the pla ⁇ mid pREP4, which expre ⁇ e ⁇ the lad repre ⁇ sor and also confers kanamycin resistance (Kan r ) .
• Transfortnants are identified by their ability to grow on LB plate ⁇ and ampicillin/kanamycin re ⁇ istant 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
• 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.
• 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 ⁇ olution for 12 hour ⁇ the protein i ⁇ dialyzed to 10 mmolar ⁇ odium pho ⁇ phate.
• the DNA sequence encoding the full length hChat protein, ATCC # 75856, is amplified using PCR oligonucleotide primers corresponding to the 5' and 3' sequence ⁇ of the gene:
• the 5' primer has the sequence 5' CGCGGGATCCACCATGAAGGCTTCCAGCCGCTTC 3' and contain ⁇ a Bam HI re ⁇ triction enzyme site (in bold) followed by 3 non-specific nucleotide ⁇ and 21 nucleotide ⁇ encoding hChat which resembles an efficient signal for the initiation of tran ⁇ lation in eukaryotic cell ⁇ (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 ⁇ equence ⁇ 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 endonuclease ⁇ Bam Hi and A ⁇ p7l8 and then purified as described above. Thi ⁇ fragment i ⁇ de ⁇ ignated F2.
• the vector pA35 (modification of pVL94l vector, di ⁇ cussed 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 expres ⁇ ion vector contain ⁇ the ⁇ trong polyhedrin promoter of the Autographa californica nuclear polyhedro ⁇ is virus (AcMNPV) followed by the recognition ⁇ ites for the restriction endonuclea ⁇ e ⁇ Bam HI and A ⁇ p718.
• 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 sequence ⁇ are flanked at both ⁇ ide ⁇ by viral ⁇ equence ⁇ for the cell-mediated homologous recombination of cotransfected wild-type viral DNA.
• 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 enzyme ⁇ Bam Hi and A ⁇ p718 and then depho ⁇ phorylated u ⁇ ing 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 de ⁇ ignated 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 ⁇ g of the plasmid pBac hChat are cotransfected with 1.0 ⁇ g of a commercially available linearized baculovirus ("BaculoGoldTM baculovirus DNA", Pharmingen, San Diego, CA.) using the lipofection method (Feigner et al. Proc. Natl. Acad. Sci. USA, 84:7413-7417 (1987)).
• BaculoGoldTM virus DNA and 5 ⁇ g of the plasmid pBac hChat are mixed in a sterile well of a microtiter plate containing 50 ⁇ l of serum free Grace's medium (Life Technologies Inc., Gaithersburg, MD) .
• plaque assay performed similar a ⁇ described by Summers and Smith (supra) .
• an agarose gel with "Blue Gal” (Life Technologies Inc., Gaithersburg) is used which allow ⁇ an easy isolation of blue ⁇ tained plaque ⁇ .
• a detailed de ⁇ cription of a "plaque a ⁇ say” can also be found in the user's guide for insect cell culture and baculovirology distributed by Life Technologies Inc., Gaithersburg, page 9- 10) .
• Sf9 cell ⁇ are grown in Grace' ⁇ medium ⁇ upplemented with 10% heat-inactivated FBS.
• the cells are infected with the recombinant baculovirus V-hChat at a multiplicity of infection (MOD of 2.
• MOD multiplicity of infection
• the medium is removed and replaced with SF900 II medium minus methionine and cysteine (Life Technologies Inc., Gaithersburg). 42 hours later 5 ⁇ Ci of 35 S-methionine and 5 ⁇ Ci 35 S 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.
• the expres ⁇ ion 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.
• 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 de ⁇ cribed a ⁇ follow ⁇ :
• the DNA ⁇ equence encoding hChat, ATCC# 75856, i ⁇ constructed by PCR on the original EST cloned using two primers: the 5' primer 5' CG ⁇ MGATCCACC-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' contain ⁇ complementary sequence ⁇ to an Xba I site (bold) , translation stop codon, HA tag and the la ⁇ t 18 nucleotide ⁇ of the hChat coding ⁇ equence (not including the ⁇ top codon) .
• 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 re ⁇ triction 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 tran ⁇ formant ⁇ and examined by re ⁇ triction analy ⁇ i ⁇ for the pre ⁇ ence of the correct fragment.
• COS cells are transfected with the expres ⁇ ion vector by DEAE- DEXTRAN method (J. Sambrook, E. Fritsch, T. Maniati ⁇ , Molecular Cloning: A Laboratory Manual, Cold Spring Laboratory Pre ⁇ , (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 35 S-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 ⁇ pecific monoclonal antibody. Protein ⁇ precipitated are analyzed on 15% SDS-PAGE gels.
• RIPA buffer 150 mM NaCl, 1% NP-40, 0.1% SDS, 1% NP-40, 0.5% DOC, 50mM Tris, pH 7.5
• RNAzolTM B ⁇ ystem Biotecx Laboratories. Inc. 6023 South Loop East, Houston, TX 77033
• About 10 ⁇ g 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 Pre ⁇ , (1989)).
• the labeling reaction i ⁇ done according to the Stratagene Prime- It kit with 50ng DNA fragment.
• the labeled DNA i ⁇ purified with a Select-G-50 column.
• ADDRESSEE CARELLA, BYRNE, BAIN, GILFILLAN,
• GAGTGGGCCC ACACCAAGCA GCTGGTGGAT GAGTTTCAGG CCTCAGGAGG TGTAGGGGAG 180
• Val Arg Ser lie Gin Lys Ser lie Phe Thr Val Cys Leu Asp Ala
• Lys Leu Ser Pro A ⁇ p Ala Phe lie Gin Met Ala Leu Gin Leu Ala

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