AU4597593A - Sequence of human dopamine transporter cDNA - Google Patents

Description

SEQUENCE OF HUMAN DOPAMINE TRANSPORTER CDNA

RELATED APPLICATIONS

This application is a Continuation-In-Part of U.S. Patent Application Serial Number 07/762,132, filed September 20, 1991, which is hereby incorporated in its entirety by reference.

BACKGROUND OF THE INVENTION Field of the Invention

The invention relates to a cloned cDNA which encodes the human dopamine transporter protein. The cloned cDNA provides a means of expressing human dopamine transorter protein in a variety of contexts and also provides a means of diagnosing and treating diseases presenting abnormal expression of dopamine transporter protein.

Description of the Related Art

Throughout this application, reference is made to articles of the scientific literature and the like.

The entire content of such citations is hereby incorporated by such reference. The dopamine transporter that acts to take released dopamine back up into presynaptic terminals has been implicated in several human disorders. Cocaine binds to the dopamine transporter and blocks dopamine reuptake in a fashion that correlates well with cocaine reward and reinforcement (M.C. Ritz et al., Science 237, 1219 (1987)). Neurotoxins that cause Parkinsonian syndromes are concentrated in dopaminergic neurons by this transporter (S.H. Snyder and R.J. D'Amato, Neurology 36, 250 (1986) ; G. Uhl, Eur. J. Neurol. 30, 21 (1990)). Binding to the dopamine transporter is altered in brains of patients with Tourette's syndrome (H.S. Singer et al., Ann. Neurol. 30, 558 (1991)). These clinical links enhance interest in the structure and function of the human dopamine transporter (HUDAT) . Vulnerability to these disorders may have genetic components (E.J. Devor and C.R. Cloninger, Annu. Rev. Genet. 23, 19 (1989) ; D. Pauls and J. Leckman, New Eng. J. Med. 315, 993 (1986); R. Pickens et al., Arch. Gen. Psychiatry 48, 19 (1991)); thus identification of linkage markers for the human DAT is also of interest. Dopamine transporters act to terminate dopaminergic neurotransmission by sodium- and chloride- dependent reaccumulation of dopamine into pre-synaptic neurons (L.L. Iversen, in Handbook of Psychopharmacology, L.L. Iversen, S.J. Iversen, & S.H. Snyder, Eds. (Plenum, New York, 1976), pp. 381-442; M.J. Kuhar and M.A. Zarbin, J. Neurochem. 31, 251 (1978); A.S. Horn, Prog. Neurobiol. 34, 387 (1990)).

Cocaine and related drugs bind to these transporters in a fashion that correlates well with their behavioral reinforcing and psychomotor stimulant properties; these transporters are thus the principal brain "cocaine receptors" related to drug abuse (M.C. Ritz, R.J. Lamb, S.R. Goldberg, M.J. Kuhar, Science 237,1219 (1987); J. Bergman, B. K. Madras, S. E. Johnson, R. 0. Spealman, J. Pharmacol. Exp. Ther. 251,

150 (1989)). The transporters accumulate neurotoxins with structural features resembling dopamine; their ability to concentrate the parkinsonism-inducing toxin MPP⁺ (l-methyl-4-phenylpyridinium) is key to this agent's selective dopaminergic neurotoxicity (S.H.

Snyder, and R. J. D'Amato, Neurology 36(2), 250 (1986);

S. B. Ross, Trend. Pharmacol. Sci. 8, 227 (1987)).

Studies of the dopamine transporter protein suggest that it is an 80 kDa glycoprotein, but have not yet yielded protein sequence data (D.E. Grigoriadis, A.A.

Wilson, R. Lew, J.S. Sharkey & M.J. Kuhar, J. Neurosci.

9, 2664 (1989)). Binding of cocaine analogs such as

[³H]CFT to membranes prepared from dopamine-rich brain regions reveals two sites with differing affinities (F.

Javory-Agid, and S.Z. Langer, Naunyn-Schmiedeberg's

Arch. Pharmacol. 329, 227 (1985); J.W. Boja, and M.J.

Kuhar, Eur. J. Pharmacol. 173, 215 (1989); B.K. Madras et al., Mol. Pharmacol. 36, 518 (1989); M.J. Kuhar et al., Eur. J. Neurol. 30(1), 15 (1990); M.C. Ritz, E.J.

Cone, M.J. Kuhar, Life Sci. 46, 635 (1990).; D.O.

Calligaro, and M.E. Eldefrawi, J. Pharmacol. Exp. Ther.

243, 61 (1987); B.K. Madras et al., J. Pharmacol. Exp.

Ther. 251(1), 131 (1989); M.C. Ritz et al., J. Neurochem. 55, 1556 (1990)).

Recent elucidation of cDNAs encoding dopamine transporters from experimental animals (B. Gros et al.,

FEBS Lett. 295, 149 (1992); J.E. Kilty et al.. Science

254, 578 (1991); S. Shimada et al.. Science 254, 576 (1991); T.B. Usdin et al., Proc. Natl. Acad. Sci. USA

88, 11168 (1991) provides hybridization probes useful for isolation of their human cognate. Summary of the Invention

Described herein is a cDNA (pcHUDAT) , which encodes the human dopamine transporter protein (HUDAT) . Also described are unique features of the nucleotide sequence of the pcHUDAT predicted for its encoded mRNA and protein, restriction fragment length polymorphisms (RFLPs) and Variable Number Tandem Repeats (VNTRs) identified by this cDNA and estimates of race-specific population frequencies of these RFLPs and VNTRs. By virtue of its representation of the human dopamine transporter sequence, the pcHUDAT is advantageous over those clones isolated from other species in that better ^'results in applications having a human context would be expected. The cDNA encoding the human dopamine transporter protein (HUDAT) provides a means for diagnosing and treating disorders that arise by expression of abnormal amounts of or dysfunctional dopamine transporter molecules in a human being. It is one object of the invention to produce a cDNA that encodes the human dopamine transporter protein, a product of dopaminergic neurons that binds dopamine, cocaine and cocaine analogs and will transport dopamine and MPP+ into mammalian cells expressing it on their surface. It is a further object of the invention to utilize the cDNA to produce cell lines that express human DAT on their surface and to provide a method for the screening of compounds that influence the binding and/or transport of dopamine or cocaine or functional analogs thereof to (into) the cells. Such cell lines may also find therapeutic application for treatment of diseases caused by depletion of cell populations which normally provide for uptake of dopamine. A third object of the invention is to provide diagnostic means for assessing HUDAT expression in patients by DNA- or antibody- based tests and for assessing the onset or progression of disease by assay of HUDAT degradation.

These and other objects are accomplished by providing a cDNA encoding the dopamine transporter protein and a purified polypeptide conferring upon cells the phenotype of dopamine uptake from the surrounding extracellular medium. Further, the invention is embodied in cell lines, created by stable transformation of cells by a vector encoding the dopamine transporter protein, expressing the dopamine transporter protein on their surface. Another aspect of the invention relates to a method of using such lines to screen pharmaceutical compositions for their ability to inhibit the binding of dopamine, cocaine or analogs of these compounds to the transporter protein. Such a screening can also be accomplished by use of cells transiently expressing dopamine transporter cDNA.

The invention also relates to diagnostic applications of the dopamine transporter cDNA and anti-human DAT antibodies and to therapeutic applications of the HUDAT cDNA.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 (SEQ. ID. NO. 1) shows the nucleotide sequence of the pcHUDAT cDNA encoding the human dopamine transporter protein; the sequence is a composite derived from the sequence of clones pHCDAT2, pHCDAT3 and pHCDAT7.

Figure 2 shows the sequences of the repeat elements in the 3' untranslated portion of the pcHUDAT cDNA. Also shown is the consensus sequence of the repeats.

Figure 3 shows a comparison of the amino acid sequence of the human dopamine transporter (Hdat) protein with the amino acid sequence of the rat DAT (Datl) and also with the sequences of the human norepinephrine transporter (Hnat) and of the human gamma-amino-butyric acid transporter (Hgabat) .

Figure 4A shows a representative RFLP analysis of human genomic DNA from nine unrelated individuals digested with TaqI and hybridized with the insert portion of the pHCDAT7 plasmid. Figure 4B shows the same DNA, but hybridized with the Taq492 probe, which corresponds to nucleotides 301-793 of the pcHUDAT sequence.

DETAILED DESCRIPTION OF THE INVENTION

For many of the applications described in the examples below subfragments or variants of the HUDAT protein disclosed in the present application wherein the original amino acid sequence is modified or changed by insertion, addition, substitution, inversion or deletion of one or more amino acids are useful so far as they retain the essential binding or transport specificity for dopamine, cocaine, or functional analogs thereof. Thus, such variants of the HUDAT are considered to fall within the scope of the present invention. Such variants are easily produced by mutagenic techniques well developed in the art of genetic engineering.

Expression of heterologous proteins in E. coli is often utilized as a means of obtaining large quantities of a polypeptide. The product is an unglycosylated protein, which may be made as insoluble "inclusion bodies" in the bacterial cells. Alternatively, some proteins can be secreted into the perplasmic space by fusion to a leader sequence that directs the secretion of the translation product. Other useful fusion sequences are those which allow affinity purification of the product, such as the pGEX system (Pharmacia) , which allows purification by use of a glutathione- Sepharose column.

The promoter to be employed is dependent upon the particular protein to be expressed. Some proteins are not detrimental to the physiology of the bacteria and may be expressed using a high-level constitutive promoter. Others are somewhat toxic and so are best expressed from an inducible promoter which keeps synthesis of the heterologous protein repressed until growth of the culture is complete. The promoter is then switched on and the heterologous protein is produced at a high level.

Other considerations in bacterial expression include the use of terminator seqences in the transcription unit and the use of sequences in the 5'- untranslated portion of the mRNA to abolish secondary structure which might impede translation. Also the choice of bacterial strain can be important. Some heterologous proteins are susceptible to proteolytic degradation and so are best expressed in strains of bacteria which lack proteolytic functions. Also, strains of bacteria other than E. coli are often useful as hosts for expression systems. The best-developed alternative currently being Bacillus strains. Expression of proteins in bacteria is well- reviewed in "Current Protocols in Molecular Biology", which is published with quarterly updates by Wiley Interscience. Expression of "foreign" proteins in mammalian cells can be accomplished in two general fashions. Transient expression refers to the creation of a pool of transfected cells which harbor plasmids that are not stably maintained in the cell and so are gradually diluted out of the population. Transient expression is by nature a short term method. For reproducible expression of a heterologous protein, stable expression systems are preferable. The current state of this art includes a variety of vector systems; both integrative and autonomous vectors are available. Inducible expression of heterologous proteins in mammalian cells is difficult to achieve at the current time. Some systems have been described, but they are not yet in general use. More commonly used are vectors bearing moderate to high- level constitutive promoters. Plasmid vectors are relatively easy to use. Retroviral vectors, which rely upon packaging into infective viral particles and integration into the host cell chromosome are more difficult to use, due to the extra steps involved in creating the recombinant viruses and cell lines which secrete them, but have the advantage that they effectively introduce exogenous DNA into human cell lines. Vaccinia virus vector systems are also in widespread use. Other viral vectors are under development for gene therapy systems, including adenovirus-derived vectors.

The preferred embodiments of the invention are described by means of the following examples. These examples are intended to be illustrative, rather than limiting in scope. It is understood that variations in the materials and techniques described below will be apparent to those skilled in art and such are to be considered to fall within the scope and spirit of the instant application.

Example 1 Isolation and sequencing of cDNA encoding human dopamine transporter

To isolate human cDNAs for the dopamine transporter, cDNA libraries prepared from "substantia nigra" and "brainste " dissections containing cells known to express the transporter were screened with hybridization probes prepared from the rat cDNA, pDATl (S. Shimada et al.. Science 254, 576 (1991)). Sequences from the 3' untranslated region of the rat cDNA were not used because of the presence of CA dinucleotide repeats. Human brain stem and substantia nigra cDNA libraries (Stratagene, La Jolla, CA) were plated and blotted onto duplicate replica nitrocellulose (Schleicher and Schuell, Keene, NH) filters, which were incubated for 1 hour at 37°C with proteinase K (50 μg/μl in 2 x SSPE/0.1%SDS) to reduce filter background, washed in 5 x SSC/0.5% SDS/lmM EDTA, prehybridized and hybridized at 42°C, and washed at 54°C in 0.4 x SSC/0.5% SDS. The hybridization probe was a 2300 bp Eco RI fragment of the rat dopamine transporter CDNA6 (S. Shimada et al., Science 254, 576 (1991)) [³²P] labeled by random priming (Prime It kit, Boehringer Mannheim), and hybridized at approximately 10⁶ cpm/ml. Positively-hybridizing cDNA clones were purified from the brainstem library, autoexcized according to protocols provided by the manufacturer (Stratagene) , and termed pHCDAT2, pHCDAT3, and pHCDAT7. Sequencing was performed on an Applied Biosystems automated sequencer as described (S. Shimada et al.. Science 254, 576 (1991)) . Sequence analysis was performed using the GCG software package (J. Devereaux, et al., Nucleic Acids Res. 12, 387 (1984)).

Screening of more than 2 x 10⁶ plaques from the substantia nigra library produced no positives. Screening 1 x 10⁶ plaques from the brainstem library yielded 11 positively-hybridizing plaques, three of which were identified as human DAT clones by sequence analysis. These clones were identified as representing the 5'-half (pHCDAT2, bases 1-1733), the 3'-half (pHCDAT3, bases 1679-3919), and an internal portion (pHCDAT7, bases 653-1434) of the human DAT cDNA whose reconstructed full-length sequence is shown in Figure 1. The structure of this cDNA resembles the structure of the rat cDNA DAT1, with a modest 5' untranslated region and a long 3' untranslated region. Both 5' and 3' untranslated regions are longer than those of the rat cDNA pDATl, however, making the length of the predicted human mRNA greater than the 3.7 kb observed for the rat mRNA (S. Shimada et al., Science 254, 576 (1991)). A striking difference between rat and human cDNAs is found in the 3' untranslated region where the human cDNA displays 10 copies of a 40 bp repetitive element that are arrayed in head-to-tail fashion and are absent from the rat cDNA (Figs 1,2). These elements are highly stereotyped. The sequence of each element is more than 90% identical to the consensus sequence listed at the bottom of Figure 2, although the seventh repeat displays a 5 base pair insertion from its 24th to 28th nucleotides. The consensus element found here is 68% G+C. No exact match is found in searches of the EMBL/genbank data base, release 70. However, sequences conferring up to 70% nucleic acid identity over up to 37 of these bases are found in viral sequences, especially with herpesvirus sequences (e.g. locus HS1US) .

The open reading frame predicted by the HUDAT cDNA encodes 620 amino acids, identical in size to the rat DATl cDNA except for an additional amino acid (199) not found in the rat sequence (Fig. 3) . This open reading frame predicts amino acid sequences that are 94% identical to those encoded by the rat dopamine transporter cDNA (S. Shimada et al.. Science 254, 576 (1991)). This high degree of conservation, and the weaker identities with the human norepinephrine and GABA transporter cDNA (H. Nelson et al., FEBS Lett. 269, 181-184 (1990); T. Pacholczyk et al.. Nature 350, 350-354 (1991)) (Fig. 3), identifies this as the human homolog of the rat DATl.

The amino acid sequence predicted by the HUDAT cDNA reveals interesting differences from the rat cDNA. It lacks one of the 4 consensus sites for N-linked glycosylation noted in the rat DATl cDNA (Fig 3, + symbol) . Three adjacent amino acids distinguish the human from the rat proteins at this locus; no other portion of the molecule differs by this extent.

Human DAT amino acids predicted to lie within hydrophobic, putative transmembrane domains show 97% amino acid identity between the rat and human transporter cDNAs. This conservation is higher than the 87% conservation in regions thought not to span the membrane, and is consistent with the high conservation in these regions among different sodium dependent transporter family members. The most striking difference between the rat and human transporters occurs in the putative second extracellular domain, at which each of the transporters cloned to date displays consensus sites for N-linked glycosylation. The glycosylation of the rat dopamine transporter has been defined in biochemical studies that suggest 20 to 30 kd of the molecular weight of the mature protein may consist of sugar (R. Lew et al.. Brain Research 539, 239 (1991) . Four potential N-linked glycosylation sites indicated in the rat transporter contain classic asparagine - X - serine/threonine sequences. Three of these sites are conserved among the rat and human sequences, but a middle glycosylation site, potentially the most distant from the embedding membrane, is absent in the human transporter. The amino acids surrounding this site provide the largest area of amino acid sequence divergence between the rat and human transporters. If glycosylation is evenly distributed among the different potential sites for N-linked glycosylation, these observations would predict that the human dopamine transporter might display less glycosylation than the rat, and that its molecular weight might be correspondingly smaller. The function of the glycosylation has not been identified to date; changes in ligand recognition, membrane targeting of the molecule, or even in cell/ cell recognition might conceivably result from these differences in glycosylation. The repeated motifs in the 3' untranslated regions of these cDNAs are another interesting difference from the rat sequence. Smaller polymorphic repeated elements have gained recent attention due to their implication in the fragile X syndrome and myotonic dystrophy (J.D. Brook, et al., Cell 68, 799-808 (1992) ; Y. Fu et al., Cell 67, 1047-1058 (1991) ; V.A. McKusick, Mendelian Inheritance in Man. 9th edn. , Johns Hopkins University Press, Baltimore, 1990, 2028 p.). The rat sequence does demonstrate 25 copies of a small dinucleotide CA repeat from bases 2476 to 2525 of the 3' untranslated region of its mRNA; CA repeats are absent from the human cDNA (S. Shimada et al., Science 254, 576-578 (1991)). The sequence of the longer hDAT repeated element is not found the rat cDNA, nor in searches of other sequences found in databanks. The significance of the partial matches in viral genomes is unclear. These repeated elements might alter mRNA properties, perhaps including secondary structure and/or half-life, in ways that could contribute to the regulation of this gene's expression. Search of this sequence using the stemloop program yields more than 150 possible loops with as many as 18 stabilizing hydrogen bonds. Conceivably, population variants in the number of these repeats could also contribute to heterogeneity in DAT function.

Example 2 Restriction Fragment Length Polymorphism (RFLP) analysis DNA was obtained from leukocytes, digested with TaqI. and analyzed by Southern blotting using pHCDAT7 as the initial hybridization probe. Simpler patterns were also obtained using two other hybridization probes. Taq 120 corresponds to bases 668 to 787 of the HDAT (see below) , and was generated by hybridizing 65 and 72 base oligonucleotides of opposite sense and extending the product using large fragment of DNA polymerase I and [³²P]-dCTP or by random priming of these two hybridized oligonucleotides, as described (A. Feinberg and B. Vogelstein, Analyt. Biochem. 132, 6-9 (1982); S. Shimada et al.. Science 254, 576-578 (1991)). Identical results were also obtained using a random-priming labeled 492 base pair cDNA fragment (Taq 492) corresponding to bases 301 to 793 of this sequence. Probes were hybridized to filters containing DNA from unrelated individuals at 42°C in hybridization solution containing 50% formamide as described. Identical results were obtained with final washes at 68°C in 0.2 X SSC/0.2% SDS or at 54°C in 0.4 X SSC/0.5% SDS. Patterns from these Southern blots were analyzed by two independent observers.

Digestion of DNA from 20 unrelated individuals with nine different restriction endonucleases revealed Southern blot patterns in each case that were consistent with the presence of a single gene. There were no clear interindividual differences in Southern blot restriction patterns using radiolabeled pHCDAT 7 after digestion with Alu I, Bam HI, Eco RI, Hae III, Hind III, Msp I, and Rsa I. Three other enzymes, Pst I, Hinf I and Taq I revealed polymorphisms. We focused on the polymorphisms identified by Taq I. When probed with radiolabeled pHCDAT 7, more than six bands were obtained from Tag I restricted DNA, many of which showed polymorphic patterns (Fig 4A) . Hybridization survived washes of up to 68°C, consistent with specificity. A simpler pattern was revealed when hybridization was performed with the Taq 120 hybridization probe or with the cDNA hybridization probe Taq 492 (Fig. 4B) . Two hybridizing bands of 7 and 5.6 kilobases were observed and termed Al and A2. Taq I Al and A2 RFLP frequencies are presented the Table. Of 272 chromosomes from 136 individuals examined 36% showed the Al form, 64% showed the A2 form. There was a significant racial dimorphism in these distributions such that 26% of Caucasians, but 42% of blacks displayed the Al RFLP (χ²=7.45, p< 0.01). The rich patterns of Taq I RFLPs identified with this cDNA sequence could relate to the fact that the clone itself contains three sites for Taq I cleavage. Further studies are thus likely to detect other polymorphisms, because extreme variability of bands in the initial Taq I restriction digestions has already been documented.

The tandem repeat in the 3'region of this gene also provides a Variable Number Tandem Repeat (VNTR) . The means for examining the distribution of alleles of the VNTR is set forth at the end of Example 3 below.

The hybridization probes that we have described provide useful markers for linkage analysis that would help to exclude the regions around the dopamine transporter gene from involvement in familial disorders. Human dopamine systems are involved in a number of human disorders, with specific implication of involvement of transporter mechanisms in psychostimulant abuse, Parkinsonism, and Tourette's syndrome (E.J. Devor and C.R. Cloninger, Annu. Rev. Genet. 23, 19-36 (1989) ; D. Pauls and J. Leck an, New Eng. J. Med. 315, 993-997 (1986); R. Pickens et al.. Arch. Gen. Psychiatry 48, 19-28 (1991); M.C. Ritz et al.. Science 237, 1219-1223 (1987); S. Shimada et al., Science 254, 576-578 (1991); H.S. Singer et al., Ann. Neurol. 30, 558-562 (1991); S.H. Snyder S.H. and R.J. D'Amato, Neurology 36, 250-258 (1986); G. Uhl, Eur. J. Neurol. 30, 21-30 (1990)). The human dopamine transporter cDNAs and RFLP information described here should provide useful tools to study its possible role in these and other human disorders. Example 3 (Predictive) A genetic component of substance abuse behavior identified bv RFLP analysis of the human DAT gene

Abuse of substances, including drugs and alcohol, is currently viewed as arising from a combination of biological, psychological, and social factors (J.S. Searles, J Abnorm Psychol. 97,153-167 (1988); E.J. Devor and C.R. Cloninger, Annu Rev Genet. 23, 19-36 (1989) ; K.R. Merikangas, Psychological Medicine 20, 11-22 (1990)). Genetic contributions to susceptibility to alcoholism are supported by family, twin, and adoption studies. (D.S. Goodwin, Arch Gen Psychiatry 36, 57-61 (1979); C.R. Cloninger et al.. Arch Gen Psychiatry 38, 861-868 (1981) ; C.R. Cloninger, Science 236, 410-416 (1987)). A genetic component of vulnerability to drug abuse has also been suggested in both twin and adoption studies (R.J. Cadoret et al.. Arch Gen Psychiatry 43, 1131-1136 (1987); R.W. Pickens et al., Arch Gen Psychiatry 48, 19-28 (1991)). A number of substances which share the potential for abuse by humans also share the ability to enhance dopamine activity in mesolimbic/mesocortical circuits thought to be important for behavioral reward and reinforcement (A.S. Lippa et al., Pharmacol Biochem Behav. 1, 23-28 (1973) ; G. Di Chiara and A. Imperato, Proc Natl Acad Sci USA 85, 5274-5278 (1988) ; R.A. Wise and P.P. Rompre, Annu Rev Psychol. 40, 191-225 (1989)). Cocaine's ability to inhibit re-uptake of dopamine, for example, points strongly toward a possible direct action for this highly-reinforcing drug in these dopaminergic circuits (M.C. Ritz et al.. Science 237, 1219-1223 (1987); D.E. Grigoriadis et al., J Neurosci. 9, 2664-2670 (1989)) . Blum, Noble and co-workers first reported that the "Al" TaqI restriction fragment length polymorphism (RFLP) of the human dopamine D₂ receptor gene (DRD2, D.K. Grandy et al.. Am J Hum Genet. 45, 778-785 (1989)) was associated with alcoholism (K. Blum et al., JAMA 263, 2055-2060 (1990)); 69% of alcoholics displayed this RFLP compared to 20% of non-alcoholics. 42% of 504 Caucasian alcoholic individuals reported in literature to date display this RFLP, while only 27% of 461 Caucasian "control" individuals are Al positive (G.R. Uhl et al.. Arch Gen Psychiatry 49, 157-160 (1992); E. Turner et al., Biol Psychiatry 31, 285-290 (1991) . These data come from eight previous studies, five of which find significant associations between RFLP and alcoholism, and provide evidence for a significant association between gene markers and behavior.

Examination of gene marker/behavior associations in drug abusers raises several methodological concerns. Relatively few individuals who abuse drugs abstain from alcohol, and many individuals who use drugs often self-administer multiple substances (D.R. Wesson et al. , eds. Polydrug Abuse: The Results of a National Collaborative Study. New York, NY: Academic Press, Inc. ; 1978) . Drug-using populations may also differ from one another and from the general population in racial, ethnic and other features that might be associated with altered distributions of the alleles for different genes (M.R. Gillmore et al., Am J Drug Alcohol Abuse 16, 185-206 (1990)). Also, some clinical assessments may not focus on the heritable features of the disorder (R.W. Pickens et al.. Arch Gen Psychiatry 48, 19-28 (1991)). A study of D₂ dopamine receptor gene markers in polysubstance users and control subjects provides a useful model for investigating the association between alleles of the DAT gene and substance abuse behaviors or other behavioral disorders such as Tourette's syndrome. We have investigated the 3' TagI Al RFLP examined in previous studies of alcoholics, and a more 5' TaqI RFLP ("B") located closer to regulatory and structural/coding regions of the gene (X.Y. Hauge et al., Genomics 10, 527-530 (1991)). Only Caucasian individuals were included in this study because of evidence for different distributions of TaqI A and B markers in white and black individuals (Dr Bruce O'Hara et al, unpublished data). Substance users were identified according to two approaches. One group of users met criteria for lifetime DSM-III-R (Diagnostic and Statistical Manual of Mental Disorders. Revised Third Edition. Washington, DC: American Psychiatric Association; 1987) psychoactive substance use disorder(s) . A second group of users was identified based on their peak lifetime use of psychoactive substances. This quantity-frequency approach was chosen because of evidence that heavy use of alcohol may display significant heritability in males and females (R.W. Pickens et al., Arch Gen Psychiatry 48, 19-28 (1991)). Control subjects were free of significant lifetime substance use.

i) Subject Recruitment: 288 Caucasian substance-using and control subjects were recruited from three sources; 21% were female. 224 drug-using and control volunteers consenting to research protocols at the Addiction Research Center (ARC) in Baltimore, Maryland were studied. The ARC is the major federal drug abuse research facility that recruits through advertisement and word of mouth for participation in treatment and non-treatment studies. 12 volunteers from a chronic hemodialysis unit on the same campus, both users and controls, augmented this sample. A third group of users consisted of 52 HIV seronegative participants in an ongoing east Baltimore study of HIV infections in intravenous drug users (D. Vlahov et al., Am J Epid. 132, 847-856 (1990)). Each subject was individually interviewed to elicit information characterizing substance use. 192 users and 56 controls were assessed according to a quantity-frequency approach. 137 users met criteria for DSM-III-R psychoactive substance use disorders. 97 users received both assessments. Written informed consent was obtained from all subjects.

Quantitv-Freguency Approach: Trained interviewers assessed subjects with the Drug Use Survey (DUS) interview (see below) in a confidential setting. The amount, frequency, and/or dollar cost at the time of lifetime peak use were recorded for each of 15 different psychoactive drugs or drug classes used more than five times. Blinded ratings of lifetime peak use of each individual substance were made on a four-point scale: 0=absent, l=minimal, 2=moderate, or 3=heavy use as indicated in Table I. A composite "Total Use" index was constructed from the pooled ratings of use of all individual substances as follows: "0" = up to minimal use of alcohol, marijuana, or nicotine and no use of other drugs; "1" = moderate use of alcohol or nicotine and/or minimal use of other drugs; "2" = heavy use of alcohol or nicotine, moderate use of marijuana, and/or up to moderate use of other drugs; "3" = heavy use of any illicit drug. Thus, neither heavy use of alcohol or nicotine was sufficient to confer a rating of heavy total drug use. Control subjects were identified as those individuals with Total Use scores of 0 or 1; substance abusers were individuals with Total Use scores of 2 or 3.

DSM-III-R Diagnoses: Trained interviewers administered the Diagnostic Interview Schedule Version III Revised (DIS-III-R, L.N. Robins et al..NIMH Diagnostic Interview Schedule Version III Revised (Version 11/7/89) . Department of Psychiatry, Washington University School of Medicine, St. Louis, MO.) to provide lifetime DSM-III-R diagnoses of psychoactive substance use disorders including nicotine and alcohol. Reliability and Validity of Drug Use Information: Drug Use Survey (DUS) ratings were evaluated in subjects who were: (a) assessed with the DUS on two different occasions at 3 to 13 months apart (n=31) , (b) tested for lifetime DSM-III-R psychoactive substance use disorders by the DIS-III-R²³ (n=18) and the Structured Clinical Interview for DSM-III-R (R.L. Spitzer et al. , Structured clinical interview for DSM-III-R - patient version (with psychotic screen) - SCID-P (W/Psvchotic Screen) - 5/1/89) . Biometrics Research Department, New York State Psychiatric Institute, New York, New York) (SCID; n=17) , and (c) checked for urinary excretion of psychoactive drugs and metabolites on the day of the DUS (n=56) . For the 18 DIS-III-R-assessed subjects and the 17 SCID-assessed subjects, DUS ratings were completed without knowledge of psychiatric assessment information. Genotypes were not available for 17 subjects assessed with the SCID and were not included in the genetic analyses. Table I. Drug Use Survey - Rating Criteria

Substance

Cigarettes 0 = never smoked cigarettes

1 = 1 to 15 cigarettes per day

2 = 16 to 25 cigarettes per day

3 = more than 25 cigarettes per day

Alcohol 0 = never used alcohol

1 = up to 4 drinks per drinking occasion, fewer than 10 drinking occasions/month

•t = up to 4 drinks per drinking occasion, more than 10 drinking occasions/month,

OR, more than 4 drinks per drinking occasion, but fewer than 10 drinking occasions/month

= 5 or more drinks per drinking occasion, more than 10 drinking occasions/month

Heroin; 0 = never used heroin/other opiates illicitly

Other 1 — used 1 time/week or less than

$30/week

Opiates 2 = 2 to 6 times/week , spending $30 to

$100/day

3 = daily use, typically spending >

$100/day

Cocaine 0 = never used cocaine

1 = less than 2 grams per week (up to $150/week) ; typical use - about 1 gram per month

2 = 2 to 4 grams per week (more than

$150/week but less than $300/week)

3 = more than 4 grams per week, usually up to 7 to 10 grams/week; (more than

$300/week, usually much higher; daily use common) Marijuana 0 = never used marijuana

1 = up to one joint/day

2 = 2 to 3 joints per day

3 = 4 or more joints per day

Minor

Tranquilizers, 0 = never used substance

Amphetamines, 1 = fewer than 1 use per week

Barbiturates, 2 = 1 to 6 uses per week (4 to 24 uses/month)

Hallucinogens, 3 = 7 or more uses/week (more than

24 Inhalants, PCP, uses/month)

Antidepressants,

Other Tobacco products.

Other Substances

ii) DNA Extraction and Analysis: Blood was obtained in EDTA-containing evacuated sterile tubes from each subject and stored at 4°C and/or frozen at -70°C in polypropylene tubes. DNA was extracted from non-frozen samples after initial isolation of nuclei and from frozen blood by selective white blood cell sedimentation followed by standard extraction methods (J. Sambrook et al., eds. "Molecular cloning: a laboratory manual" (2nd edition) . Cold Spring Harbor (NY) Laboratory Press; 1989) . 5-10 μg of this DNA was digested with TaqI as recommended by the manufacturer, or with 20-fold excess of this enzyme for several individuals displaying A3 alleles. DNA fragments were electrophoresed using 0.8% agarose gels containing ethidium bromide at 1-2 volts per centimeter for 16 hours, transferred to nylon membranes, and immobilized by UV crosslinking.

Hybridization was performed for 16-24 hours at 42°C in 50% formamide, 5xSSC, 50 mM NaP0₄ (pH 6.8), 1% SDS, ImM EDTA, 2.5 x Denhardt's solution, 200 μg/ml herring sperm DNA, and 4X10⁶ cpm/ml of radiolabelled DNA (see below) . Washing for 20 minutes in 2XSSC at room temperature was followed by two 30 minute washes in 0.4 x SSC/0.5%SDS at 55°C. Washed blots were exposed to Kodak XAR film 1-6 days with an intensifying screen at -70°C. Band sizes were compared to λ DNA molecular weight standards, and with patterns previously defined (K. Blum et al., JAMA 263, 2055-2060 (1990); A.M. Bolos et al., JAMA 264, 3156-3160 (1990). After TaqI A RFLP status was determined, ³²P decay allowed re- hybridization of the same blots with hybridization probe for TagI B ascertainment. When background levels of radiation were not reached, filters were incubated at 65°C for 30 min in 2 mM TRIS (pH 8) , 1 mM EDTA, and 0.1% SDS to remove residual hybridized probe. RFLP status was assigned by two independent raters unaware of the clinical status of the subjects.

iii) Hybridization probes: A 1.7 kb BamHI fragment of the human genomic clone encoding the dopamine D₂ receptor (λhD2Gl) was subcloned into the BamHI site on bluescript SK+ to produce phD2-9, which was used to detect Al, A2, and A3 patterns in the Southern analyses, as described (K. Blum et al., JAMA 263, 2055-2060 (1990) ; A.M. Bolos et al., JAMA 264, 3156-3160 (1990)) (Dr Bruce O'Hara et al, unpublished data) . λhD2G2 was used to detect the TaqI "B" patterns. DNAs were radiolabelled using random priming and ³²P-CTP to specific activities of approximately 10⁹ cpm/μg (A. Feinberg and B Vogelstein, Anal Biochem. 137, 266-267 (1984)).

iv) Analyses:

a) Association analyses: A two-tailed Pearson chi square test (with Yates' correction for continuity) was used to evaluate the association between Al RFLP presence and substance use/abuse; the same analysis were repeated for the Bl RFLP. Association was first tested contrasting controls and substance users meeting criteria for any lifetime DSM-III-R substance dependence disorder. Next, controls were contrasted with substance users who had been assessed with the DUS. Data for both groups of substance-using subjects were pooled and compared to RFLP frequencies for controls, b) Comparisons with other data: Pooled TaqI Al RFLP data from ARC users was compared with values obtained for Caucasian controls in other studies. c) Subtracting heavy alcohol users: DUS-assessed users free of heavy alcohol use were compared with controls to test whether the associations observed might be attributed solely to alcohol.

TagI A and B RFLPs were assigned with 100% agreement between two independent raters.

Substance use assessment by means of the Drug Use Survey showed several features suggesting validity and reliability. For 31 subjects whose DUS was elicited twice, interrater reliability correlations for severity ratings ranged from 0.83 to 1.00 (median = 0.94), while test-retest reliability correlations for individual drugs ranged from 0.53 to 0.94 (median = 0.78). For 35 subjects with DIS-III-R or SCID assessments and independent DUS ratings, analysis of the correspondence between a positive lifetime DSM-III-R Substance Use diagnosis and moderate to heavy substance use on the DUS yielded a kappa value of 0.68 (91% agreement). Finally, drugs tested as positive in urine drug screening were reported used 84% of the time (n= 56) in the DUS assessment.

TaqI A and B RFLP frequencies for substance-using and control subjects are presented in Table II. For the TaqI Bl RFLP, a significant association was found comparing users with at least one lifetime DIS-III-R Substance Use Disorder diagnosis and DUS-assessed controls (χ² = 6.74, p < 0.01). For the TaqI Al RFLP, analysis of the same groups revealed a significant association (χ² = 3.98, p < 0.05). Comparison of DUS- assessed users to DUS-assessed controls revealed a significant association for the TaqI Bl RFLP (χ² = 5.45, p < 0.02) and a trend towards significant association for the TaqI Al RFLP (χ² = 3.14, p < 0.08). Table III presents TaqI A and B genotypes (homozygotes and heterozygotes) for DUS-assessed controls and users.

Table II. D₂ Dopamine Receptor Gene RFLPs in Users and Controls

Group Al Present (%) Bl Present (%)

I. POLYSUBSTANCE USERS a) DSM-III-R Substance Use Diagnosis b) DUS* Heavy Use (Total Use=3) c) DUS Moderate Use (Total Use=2) d) DUS Total Use 2 & 3 Combined Combined UsersQ

II. CONTROLS a) DUS Minimal Use (Total Use=l b) DUS Sparse Use (Total Use=0) Combined Controls

III. PREVIOUSLY REPORTED CONTROLS

Blum et al,¹⁵ (1990)^*

Blum et al,³⁶ (1991)^*

Comings et al,³⁵ (1991)^*

Parsian et al,³⁷ (1991)^*

Todd et al,⁺ (1991)^{^}

Bolos et al ° (1990)

Comings et al,³⁵ (1991) Gelernter et al,^3δ (1991) CEPH-Co ings et al,³⁵ (1991) Grandy et al,¹⁴ (1989) O'Hara et al,⁺⁺ (1992) subtotal 27.1 (152/560)

Combined Controls: Prior and Current Reports total ( )

"DUS = Drug Use Survey ^aThe subject total here (n=232 users) reflects 40 users who received only the DIS-III-R + 82 users who received the DIS-III-R and the DUS + 110 users who received only the DUS. Four subjects who received both the DIS-III-R and the DUS did not meet criteria for a DSM- III-R diagnosis but were classified as users (in the DUS and "Combined Users" comparisons) on the basis of their DUS ratings. ^*Screened to exclude alcohol or drug abuse. ⁺Cited in Cloninger et al., JAMA 266, 1833 (1991). ^Unpublished data.

Table III. D₂ Dopamine Receptor Gene RFLP Frequencies in Users and Controls

DUS Heavy Use DUS Moderate Use DUS Sparse Use

(Total Use=2) (Total Use=0) 4/13 = .308 2/13 = .154

26/76 = .342 6/76 = .079

37/159= .233 28/159= .176

4/9 = .444 2/9 = .222

19/59 = .322 3/59 = .051 44/180= .247 31/180= .172 denominator = total number of subjects with a given genotype (eg, Al/Al) .

No significant differences in RFLP frequencies were found between DUS-assessed substance users and DIS-III-R-assessed users (for TaqI Al, χ² = 0.005, ns; for TaqI Bl, χ² = 0.331, ns) . We thus reanalyzed the data by pooling substance users assessed in both fashions for comparison with controls. Analysis of TaqI Bl data (χ² = 6.31, p < 0.02) and TaqI Al data (χ² = 4.46, p < 0.04) again revealed significant associations.

Comparisons of TaqI Al RFLP frequencies in ARC users and controls from all other published studies revealed significant associations when the controls were assessed (χ² = 15.41, p < 0.001), unassessed (χ² = 9.31, p < 0.003), or pooled (χ² = 14.80, p < 0.001).

To examine whether the effects noted could be attributed chiefly to the extent of alcohol intake, heavy alcohol users achieving DUS alcohol ratings of 3 were eliminated from the user group and reanalysis performed. Omitting heavy alcohol users did not significantly alter the elevated frequencies of the TaqI Bl RFLP found in the user group. 32.3% of all polysubstance users and 32.9% of the 73 polysubstance users free of heavy alcohol use displayed the TaqI Bl marker.

The hypothesis that individual differences in substance abuse may be due, in part, to different dopamine D₂ receptor alleles marked by Taq I RFLPs at the gene's 3' and 5' ends arises from initial work in alcoholics (K. Blum et al., JAMA 263, 2055-2060 (1990)). The hypothesis is strengthened by a compelling biological rationale for interactions between abused drugs and brain dopamine systems (G. Di Chiara and A. Imperato, Proc Natl Acad Sci USA 85, 5274-5278 (1988) ; R.A. Wise and P.P. Rompre, Annu Rev Psychol. 40, 191-225 (1989)). In the current example, significant associations with heavy substance use or abuse were found consistently for the TaqI Bl RFLP and less consistently for the TaqI Al RFLP. These findings provide preliminary evidence that a more 5' TaqI RFLP (Bl) may represent a better marker for a DRD2 gene variant possibly predisposing carriers to heavy substance use or abuse. Selection of drug using and control populations provides opportunities for different approaches that could influence the results obtained. Many substance abusers use multiple psychoactive substances ( D.R. Wesson et al., eds. Polvdruq Abuse: The Results of a National Collaborative Study. New York, NY: Academic Press, Inc.; 1978); 71% of the 192 DUS-assessed users in the current study reported moderate to heavy use of three or more different substances. To reflect this fact, we first studied subjects who frequently use multiple drugs, attempted to characterize each drug used by each subject, and analyzed data on the basis of overall lifetime peak use. This approach might provide a weaker test of linkage if only a single abused substance displayed such genetic association. For example, if only alcohol abuse contributed to the associations noted here, we might anticipate a weaker association between Bl RFLPs and substance abuse if individuals with heavy alcohol consumption were eliminated from our sample. In fact, elimination of heavy alcohol users (DUS rating=3) resulted in no decrease in the differences between the remaining DUS-assessed drug-abusing and control individuals for the TaqI B RFLP. The characterization of these subjects also raises important issues of assessment type, validity and reliability. Errors in clinical assessment would weaken tests of the allelic association hypothesis. In addition, studying behaviors that could contribute to features of clinical diagnosis but might not reflect the behavioral impact of a DRD2 gene variant could yield false-negative results.

We originally began work with the DUS, an interview-based assessment of substance use that enabled approximate quantification of peak lifetime use for several types of substances and appeared to provide an assessment of a basic feature of substance abuse: level of substance consumption. Psychiatric genetic work using classical methods suggests that heavy substance use can show substantial genetic determinants (R.W. Pickens et al., Arch Gen Psychiatry 48, 19-28 (1991) ; C.R. Cloninger and T. Reich, In: Kety SS, Rowland LP, Sidman RL, Matthysse SW, eds. Genetics of neurological and psychiatric disorders. New York, NY: Raven Press: 1983; pp. 145-166) . Reliability and validity of the quantity-frequency approach to subjects' drug use were supported by the correlations between drug use assessments made on two occasions, assessments made with multiple instruments, and correlations with results of urine drug screens. However, several individuals who reported heavy use of various drugs did not fulfill criteria for DSM-III-R diagnoses of dependence or abuse on SCID or DIS-III-R assessments of the same drugs (S.S. Smith et al., "Validation of an instrument for quantifying drug use self-report: The ARC Drug Use Scale". Presented at the 53rd Annual Scientific Meeting, The Committee on Problems of Drug Dependence, June 16-20, 1991, Palm Beach, FL) .

We also evaluated subjects by determining lifetime psychiatric diagnoses of psychoactive substance use disorders using a structured psychiatric interview, the DIS-III-R, which can also demonstrate reliability and validity (J.E. Helzer et al.. Arch Gen Psychiatry 42, 657-666 (1985); N. Oskooilar et al., DIS Newsletter 8, 9- 10 (1991)). Comparison of TaqI A and B RFLP frequencies in substance-using subjects failed to indicate significant differences between the quantity- frequency and psychiatric diagnosis approaches. These results suggested that we could combine subjects meeting criteria for DSM-III-R Substance Use diagnsoses with subjects reporting moderate to heavy drug use. It is still conceivable. however, that behavioral effects of a gene might be differentially reflected in quantity/frequency or in disease/disorder approaches to defining the affected group. The RFLPs studied here are the result of polymorphic TaqI restriction sites in which "A" RFLPs are located ca. 9 kb 3' to the final exon of the D₂ receptor gene (0. Civelli, personal communication) and "B" RFLPs are located near the first coding exon (X.Y. Hauge et al., Genomics 10, 527-530 (1991)). These polymorphisms could have functional relevance if base pair differences directly influenced the gene's regulation. Alternatively, they could provide markers for structural or regulatory changes in other regions of the gene if these other changes and the TaqI variations were maintained together by linkage disequilibrium resulting in specific haplotypes (G.R. Uhl et al.. Arch Gen Psychiatry 49, 157-160 (1992)). This linkage disequilibrium does exist (X.Y. Hauge et al., Genomics 10, 527- 530 (1991) ; Dr. Bruce O'Hara et al, unpublished data) . In our data, for example, the expected frequency of the A2/A2-B2/B2 haplotype would be 43% based on the frequencies of the A2 and B2 allelic markers. However, the observed frequency of this haplotype was 61% (χ² = 16.33, p < 0.0001) (76% for controls and 57% for users, χ² = 5.15, p < 0.03), indicating substantial linkage disequilibrium.

The lack of strong association between D₂ receptor gene RFLPs and substance use evident in this study is consistent with estimates of the heritable components of alcoholism and drug abuse (E.J. Devor and C.R. Cloninger, Annu Rev Genet. 23, 19-36 (1989).; R.J. Cadoret et al., Arch Gen Psychiatry 43, 1131-1136 (1987)). One recent study of concordance rates for alcoholism in twin populations suggests that between 20 and 30% of the vulnerability to abuse or dependence on this substance may be genetic in origin (R.W. Pickens et al.. Arch Gen Psychiatry 48, 19-28 (1991)). Attempts to link familial alcohol susceptibility to specific chromosomal markers and patterns of inheritance in families have not been consistent with a single genetic locus (S.B Gilligan et al., Genet Epidemiol. 4, 395-414 (1987); C.E. Aston and S.Y. Hill, Am J Hum Genet. 46, 879-887 (1990)) . The strong association between a single gene RFLP and alcoholism found by Blum et al. (K. Blum et al., JAMA 263, 2055-2060 (1990)) would thus fit poorly with this extent of heritability. The large environmental influences on expression of alcoholism, and their study of unrelated individuals rather than defined pedigrees also make the strength of their findings surprising. To investigate the association between the DAT gene and substance abuse behaviors, one can make use of the variable number tandem repeat (VNTR) at the 3'-end of the mRΝA described in example 1. Alternatively the TaqI RFLP described in example 2 could be utilized. In general, examination of VNTR markers is preferred, as such markers have a larger number of alleles and hence are "more informative", i.e. VNTR markers identify more subtypes than a regular "site-no site" RFLP marker. The same methodology described above for the study of the D2 dopamine receptor gene can be employed. As shown above, particular attention must be paid to the diagnostic criteria for identifying the abuse behavior if the results are to be meaningful.

To assess frequencies of the VNTR, DΝA is obtained from leukocytes from research volunteers as described above. Genomic DΝA (40 ng) is subjected to 35 cycles of amplification using AmpliTaq DΝA Polymerase (1.25 U) and polymerase chain reaction with denaturing for 1 min at 93°C, and annealing/extension for 1 min at 72°C in buffer supplied by the manufacturer (Perkin- El er) . Oligonucleotides T3-5LOΝG (5'- TGTGGTGTAGGGAACGGCCTGAG-3', SEQ. ID. NO. 4) and T7-3aLONG (5'-CTTCCTGGAGGTCACGGCTCAAGG-3' , SEQ. ID. NO. 5) are used at 0.5 uM final concentration. Reaction products are separated by 5% polyacrylamide gel electrophoresis, and product sizes estimated by comparison to molecular weight standards (BRL) . 242 of the 254 chromosomes examined displayed either 9 or 10 copies of the 40 basepair repeat. Two chromosomes showed three copies, two showed 5 copies, three showed 7, four showed 8 and one showed 11 copies of the VNTR. Among individuals with 9 and/or 10 copies per chromosome there were racial differences in copy number frequencies. Whites displayed 30% and Blacks displayed 20% of the 9-copy variant. The 3' VNTR marker defined by 9 versus 10 copies of the 40 basepair repeat displayed no significant linkage disequilibrium with the more 5' TaqI RFLP (χ² values were 5.51 and 4.62 for White and Black subjects, respectively, with 8 degrees of freedom, p > 0.1.)

Example 4 (predictive) Expression of HUDAT protein in Escherichia coli and purification of the bacteriallv expressed protein

Any of several expression systems can be utilized to obtain HUDAT protein expression in E. coli. For example, the plasmid vector pFLAG system (International Biotechnologies, Inc., New Haven, CT) produces the polypeptide of interest attached to a short protein sequence that allows purification of the fusion protein by use of a monoclonal antibody directed against a hydrophilic, and thus surface localized, octapeptide. The open reading frame midportion of the HUDAT cDNA is obtained by digestion of the pHCDAT7 plasmid with EcoRI and purification of the insert fragment encoding the HUDAT protein by electrophoresis and elution from an agarose gel by standard techniques. Oligonucleotides having the sequences 5'-GGGTCTAGACG-3' and 5'- AATTCGTCTAGACCC-3' are annealed to form an adaptor and the adaptor is ligated to the ends of the insert DNA. The ligation product is digested with Xbal and cloned into the Xbal restriction site of the pFLAG vector (International Biotechnologies, Inc.). The appropriate E. coli host is transformed and colonies containing the HUDAT cDNA may be screened by colony hybridization using the pcHUDAT as probe. Positive clones are grown as large-scale cultures and the fusion protein is obtained in pure form by use of the monoclonal antibody affinity column as described by the manufacturer of the system, except that the elution buffer is modified by the addition of 0.5% CHAPS (3-[ (3-Cholamidopropyl)- dimethylammonio] 1-propane-sulfonate) . Authentic DAT protein lacking the FLAG octapeptide is obtained by enterόkinase cleavage of the fusion protein as described by the supplier of the FLAG system.

Example 5 (predictive) Purification of DAT from tissues or from transformed mammalian cells. As protein isolated from transformed bacterial cells lacks post-translational modifications, such as sugar additions, that occur in mammalian cells, the purification of the protein from tranformed COS cells is discussed. COS cells transformed as described in (S. Shimada et al.. Science 254, 576 (1991)) are subjected to a purification protocol as described for the purification of the GABA transporter (Radian, et al., J. Biol. Chem. 261, 15437-15441 (1987) with the modification that binding of labelled CFT is used to assay for the presence of DAT in the sample rather than labelled gamma- amino butyric acid. The protocol is modified as required to allow the isolation of DAT as a distinct protein by techniques known to a practitioner of the art.

Example 6 (predictive) Diagnosis of deficiency, mutant or overexpression of dopamine transporter bv PCR

mRNA obtained from tissue biopsy from a patient is converted subjected to quantitative reverse-transcript PCR (for example, see A. M. Wang, et al. PNAS USA 86:9717 (1989)) utilizing as primers oligonucleotides derived from the cDNA sequence of pcHUDAT. Use of the 5' 19-mer, GCTCCGTGGACTCATGTCTTC, bases 118 through 139 of Fig. 1 (SEQ. I.D. NO. 1) as the upstream primer and CACCTTGAGCCAGTGGCGG , the reverse complement of bases 1942 to 1960 of Fig. 1 (SEQ. I.D. NO. 1) as the downstream primer allows examination of the character of the protein coding region of the HUDAT mRNA. Variance in the expression level can be ascertained by comparison of product yield with a normal control. Abnormal mRNA structures can be diagnosed by observation of a product band of a length different from the normal control. Point mutants can be observed by use of primers and conditions appropriate for detection of the mismatch between the mutant and normal alleles. For example, the "reverse dot blot" procedure for screening the expression of several mutant alleles in a single experiment, which has been described for the CFTR gene, mutants of which cause cystic fibrosis (Erlich, H.A. , et al Science 252:1643 (1991). The HUDAT mRNA also contains a variable number tandem repeat element in the 3' untranslated portion of the mRNA which can be amplified for examination of an association between specific VNTR alleles and substance abuse behavior or diseases associated with expression of particular HUDAT alleles (See example 3) .

Example 7 (predictive) Use of dopamine transporter expression to incorporate as part of overexpression of a panel of dopaminergic genes to reconstruct a dopaminergic cell line for therapy in human diseases resulting from defective dopamine transporter expression.

cDNAs for the human dopamine transporter, and for tyrosine hydroxylase and aromatic ammino acid decarboxylase (DOPA decarboxylase) are transfected into cell types including COS cells as described above. Cells are cotransfected with the neomycin resistance marker, selected by growth in G418, and then tested for their ability to synthesize and accumulate dopamine. Individual subclones may be able to take up dopamine, without the ability to synthesize it. However, individual subclones are also likely to integrate several of the plasmids. If the plasmids cannot be introduced serially or together in this direction, serial edition of tyrosine hydroxylase and DOPA decarboxylase to stable cell lines already expressing the dopamine transporter stably should be employed (see above) . The ability of cells to incorporate tritiated tyrosine into tritiated dopamine is tested via HPLC analysis and radiochemical detection as described (Uhl et al. , Molecular Brain Research, 1991) , their ability to take up tritiated dopamine is performed as described in the same reference.

These same procedures are used in transfecting cells obtained from an individual with a disease state caused by defects in dopamine transporter expression, either in the amount expressed or due to expression of a defective protein, so that stable immortalized cell lines expressing human dopamine transporter could be constructed with immunologic identity to the patient. Means of controlling the replication of these cells by encapsulating them in a matrix that is not porous to cell bodies, but able to be permeated by cell processes, or by use of inhibitory growth factors, can also be employed. A third strategy, temperature sensitive cell mutants that would not divide under physiologic temperatures (e.g. temperature sensitive COS cells variants) could be used to be able to express the dopaminergic cDNA stably, in a fashion that would produce dopaminergic cells. Each of these cell types are potential candidates for use in transplantation into striatum in individuals with striatal dopamine depletion in Parkinson's disease. Alternatively, genes could be incorporated with retroviral vectors as well- known for practitioners of the art.

Example 8 (predictive) Production of variant sequences in HUDAT protein and testing of their biological function Site directed mutagenesis using olgonucleotides is used to introduce specific single- and multiple-base changes into the HUDAT cDNA that change specific amino acids in the HUDAT protein. The ability of mutant transporters to take up [³H] dopamine, [³H] MPP+,and to bind [³H] cocaine and cocaine analogues (especially [³H] CFT) is tested as described previously (S. Shimada et al.. Science 254, 576 (1991)). The Amersham mutagenesis system (version 2.1, technical bulletin code RPN1523) can be used. Initial studies of mutants of the aspartic acid residue in transmembrane domain 1, and the serine residues in transmembrane domain 7 of the rat DAT protein have revealed substantial effects on dopamine transport, and more modest effects on cocaine binding. These results document that the residues key to dopamine transport are not identical to those crucial to cocaine binding; the first transmembrane residue change of aspartic acid (residue 79) to glycine reduces cocaine binding by 10%, but reduces dopamine transport by over 95%. Mutations in the second extracellular domain in glycosylation sites help elucidate the role of glycosylation in the functions of this molecule (See also Example 9) . Selective removal of the N and C terminal intracellular and second extracellular loop, and production of chimeric molecules with replacement of these regions with the corresponding regions of the GABA transporter further confirm the molecular features of DAT that are essential for dopamine transport and cocaine binding and allow development of agents dissociating the two processes.

Example 9 (predictive)

Alteration of carbohydrate structure in the extracellular domain of the HUDAT protein

As noted in example 1, the largest difference in the structure of the proteins predicted by the human and the rat dopamine transporter dDNA sequences is the absence of one of the four consensus sites for N-linked glycosylation of the protein (See figure 3) . By virtue of their location in the same domain of the protein expected to most influence substrate binding, that is in the extracellular portion of the protein, it is of interest to investigate the contribution of the sugars to substrate binding. Site directed mutagaenesis can be performed as described for Example 8 introduce into the human DAT cDNA the asparagine residues to which N- linked sugars are attached and the remaining amino acids which constitute the glycosylation signal for that site that are found in the rat, but not the human cDNA. The result of expression of such mutant proteins can be evaluated by photo-affinity labelling of the protein and analysis by SDS-PAGE. Digestion of the protein with various glycosidases can be performed to assess the degree to which the pattern of glycosylation has been altered, as described by Lew et al. (R. Lew et al.. Brain Research 539, 239 (1991)). For instance, co pararison of the wild-type and mutant protein, both untreated and digested with N-glycanase, would should show similar sized proteins for the digested protein, but a larger protein for the untreated mutant, compared to the untreated wild-type protein if the introduction of the asparagine glycosylation signal resulted in successful incorporation of sugar into the protein at that site. More detailed information regarding the sugar structure can be obtained by exoglycosidase digestion experiments. For example, the presence of sialic acid residues in the polysaccharide can be detected by digestion with neuraminidase. The influence of the polysaccarhide structure on function of the protein is then assessed by testing the properties of the the transporter using either stably transfected cells expressing the mutant protein, or by using cells transiently expressing the mutant transporter on their surface. The means for carrying out such functional studies are described by Shimada et al. (S. Shimada et al.. Science 254, 576 (1991)).

Example 10 (predictive) Cell lines expressing HUDAT protein on the cell surface can be used to screen candidate compounds for efficacy as dopamine (or cocaine or functional analogs thereof) agonists or antagonists by evaluating the influence of the candidate compound upon the binding of dopamine (or cocaine or functional analogs thereof) to the surface of such cells. Another assay for dopamine agonist or antagonist activity is to measure the cytotoxicity to such cells of MPP* to such cells in the presence and absence of the candidate compound. Such assays are described using cells expressing the rat DAT cDNA in Shimada et al. (S. Shimada et al., Science 254, 576 (1991)) and can be applied as well to cells expressing the human DAT cDNA.

Example 11 (predictive)

Production of antibodies to HUDAT and use of same in a diagnostic test for dopaminergic cell death.

A. Production of polyclonal antibodies.

HUDAT protein obtained as described above or synthetic polypeptides of amino acid sequence derived from the HUDAT sequence are used as immunogens in an appropriate animal. The serum is obtained from the immunized animal and either utilized directly or the antibody may be purified from the serum by any commonly utilized techniques. Polyclonal antibody directed only toward HUDAT can be isolated by use of an affinity column derivatized with the immunogen utilized to raise the antibody, again using techniques familiar to one knowledgable in the art.

B. Production of monoclonal antibodies to HUDAT Monoclonal antibodies to HUDAT or to particular epitopes of HUDAT may be produced by immunization of an appropriate animal with HUDAT protein obtained as above or with peptides of amino acid sequence derived from the HUDAT amino acid sequence. Hybridoma cultures are then established from spleen cells as described by Jaffe and McMahon-Pratt (Jaffe, CL. and MacMahon-Pratt, D. J. Immunol. 131, 1987-1993 (1983)). Alternatively, peripheral blood lymphocytes may be isolated and immortalized by transformation with Epstein-Barr virus. These cells produce monoclonal antibodies, but if desired, hybridomas can then be made from the transformed lymphocytes (Yamaguchi, H. et al. Proc. Natl. Acad. Sci. 84, 2416-2420 (1987)). Cell lines producing anti-HUDAT antibodies are identified by commonly employed screening techniques. Monoclonal antibody is then purified by well known techniques from the supernatants of large-scale cultures of the antibody producing cells. C. Diagnosis of dopaminergic cell death in vivo by immunoassay of cerebrospinal fluid of a patient using anti-HUDAT antibodies.

The death of dopaminergic neurons in the brain of a patient should result in the accumulation in the cerebrospinal fluid, which bathes these cells, of membrane debris as a product of lysis of the dead cells. Other pathologic conditions, short of cell death that result in the release of DAT protein, or degraded peptide fragments of HUDAT protein into the surrounding medium can also be imagined. The cerebospinal fluid can be sampled by lumbar puncture of a patient. The presence of degradation products of HUDAT protein is detected by immunoassay, using as the primary antibody at least one of the products obtained as described above. Elevated levels of HUDAT protein detected in the cerebrospinal fluid, compared with the range seen in normal controls is indicative of Parkinsons's disease or drug-induced neurotoxicity. Alternatively, disease progression can be monitored by the assessment of HUDAT levels in serial samples from the same patient.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Uhl, George R.

Vandenbergh, David Persico, Antonio

(ii) TITLE OF INVENTION: Sequence of Human Dopamine Transporter

(iii) NUMBER OF SEQUENCES: 5

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Birch, Stewart, Kolasch & Birch

(B) STREET: 301 N. Washington St.

(C) CITY: Falls Church

(D) STATE: Virginia

(E) COUNTRY: USA

(F) ZIP: 22046-3487

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

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(D) SOFTWARE: Patentln Release #1.0, Version #1.25

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(C) CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Murphy Jr., Gerald M.

(B) REGISTRATION NUMBER: 28,977 (ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 703-241-1300

(B) TELEFAX: 703-241-2848

(C) TELEX: 248345

(2) INFORMATION FOR SEQ ID Nθ:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 3919 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(iii) HYPOTHETICAL: NO

(vi) ORIGINAL SOURCE:

(A) ORGANISM: Homo sapiens (F) TISSUE TYPE: brainstem

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 102..1961

(D) OTHER INFORMATION: /function-- "dopamine transport" /product-- "HUDAT polypeptide"

(ix) FEATURE:

(A) NAME/KEY: misc_RNA

(B) LOCATION: 2724..3117

(D) OTHER INFORMATION: /function-- "unknown" /label= VNTR_region (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:

GAATTCCCGC TCTCGGCGCC AGGACTCGCG TGCAAAGCCC AGGCCCGGGC GGCCAGACCA 60 AGAGGGAAGA AGCACAGAAT TCCTCAACTC CCAGTGTGCC C ATG AGT AAG AGC 113

Met Ser Lys Ser

1

AAA TGC TCC GTG GGA CTC ATG TCT TCC GTG GTG GCC CCG GCT AAG GAG 161 Lys Cys Ser Val Gly Leu Met Ser Ser Val Val Ala Pro Ala Lys Glu 5 10 15 20

CCC AAT GCC GTG GGC CCG AAG GAG GTG GAG CTC ATC CTT GTC AAG GAG 209 Pro Asn Ala Val Gly Pro Lys Glu Val Glu Leu lie Leu Val Lys Glu 25 30 35

CAG AAC GGA GTG CAG CTC ACC AGC TCC ACC CTC ACC AAC CCG CGG CAG 257 Gin Asn Gly Val Gin Leu Thr Ser Ser Thr Leu Thr Asn Pro Arg Gin 40 45 50

AGC CCC GTG GAG GCC CAG GAT CGG GAG ACC TGG GGC AAG AAG ATC GAC 305 Ser Pro Val Glu Ala Gin Asp Arg Glu Thr Trp Gly Lys Lys lie Asp 55 60 65

TTT CTC CTG TCC GTC ATT GGC TTT GCT GTG GAC CTG GCC AAC GTC TGG 353 Phe Leu Leu Ser Val lie Gly Phe Ala Val Asp Leu Ala Asn Val Trp 70 75 80

CGG TTC CCC TAC CTG TGC TAC AAA AAT GGT GGC GGT GCC TTC CTG GTC 401 Arg Phe Pro Tyr Leu Cys Tyr Lys Asn Gly Gly Gly Ala Phe Leu Val 85 90 95 100

CCC TAC CTG CTC TTC ATG GTC ATT GCT GGG ATG CCA CTT TTC TAC ATG 449 Pro Tyr Leu Leu Phe Met Val lie Ala Gly Met Pro Leu Phe Tyr Met 105 110 115

GAG CTG GCC CTC GGC CAG TTC AAC AGG GAA GGG GCC GCT GGT GTC TGG 497 Glu Leu Ala Leu Gly Gin Phe Asn Arg Glu Gly Ala Ala Gly Val Trp 120 125 130 AAG ATC TGC CCC ATA CTG AAA GGT GTG GGC TTC ACG GTC ATC CTC ATC 545 Lys lie Cys Pro lie Leu Lys Gly Val Gly Phe Thr Val lie Leu lie 135 140 145

TCA CTG TAT GTC GGC TTC TTC TAC AAC GTC ATC ATC GCC TGG GCG CTG 593

Ser Leu Tyr Val Gly Phe Phe Tyr Asn Val lie lie Ala Trp Ala Leu 150 155 160

CAC TAT CTC TTC TCC TCC TTC ACC ACG GAG CTC CCC TGG ATC CAC TGC 641

His Tyr Leu Phe Ser Ser Phe Thr Thr Glu Leu Pro Trp lie His Cys

165 170 175 180

AAC AAC TCC TGG AAC AGC CCC AAC TGC TCG GAT GCC CAT CCT GGT GAC 689

Asn Asn Ser Trp Asn Ser Pro Asn Cys Ser Asp Ala His Pro Gly Asp 185 190 195

TCC AGT GGA GAC AGC TCG GGC CTC AAC GAC ACT TTT GGG ACC ACA CCT 737

Ser Ser Gly Asp Ser Ser Gly Leu Asn Asp Thr Phe Gly Thr Thr Pro 200 205 210

GCT GCC GAG TAC TTT GAA CGT GGC GTG CTG CAC CTC CAC CAG AGC CAT 785

Ala Ala Glu Tyr Phe Glu Arg Gly Val Leu His Leu His Gin Ser His 215 220 225

GGC ATC GAC GAC CTG GGG CCT CCG CGG TGG CAG CTC ACA GCC TGC CTG 833

Gly lie Asp Asp Leu Gly Pro Pro Arg Trp Gin Leu Thr Ala Cys Leu 230 235 240

GTG CTG GTC ATC GTG CTG CTC TAC TTC AGC CTC TGG AAG GGC GTG AAG 881

Val Leu Val lie Val Leu Leu Tyr Phe Ser Leu Trp Lys Gly Val Lys

245 250 255 260

ACC TCA GGG AAG GTG GTA TGG ATC ACA GCC ACC ATG CCA TAC GTG GTC 929

Thr Ser Gly Lys Val Val Trp lie Thr Ala Thr Met Pro Tyr Val Val 265 270 275 CTC ACT GCC CTG CTC CTG CGT GGG GTC ACC CTC CCT GGA GCC ATA GAC 977 Leu Thr Ala Leu Leu Leu Arg Gly Val Thr Leu Pro Gly Ala lie Asp 280 285 290

GGC ATC AGA GCA TAC CTG AGC GTT GAC TTC TAC CGG CTC TGC GAG GCG 1025 Gly lie Arg Ala Tyr Leu Ser Val Asp Phe Tyr Arg Leu Cys Glu Ala 295 300 305

TCT GTT TGG ATT GAC GCG GCC ACC CAG GTG TGC TTC TCC CTG GGC GTG 1073 Ser Val Trp lie Asp Ala Ala Thr Gin Val Cys Phe Ser Leu Gly Val 310 315 320

GGG TTC GGG GTG CTG ATC GCC TTC TCC AGC TAC AAC AAG TTC ACC AAC 1121 Gly Phe Gly Val Leu lie Ala Phe Ser Ser Tyr Asn Lys Phe Thr Asn 325 330 335 340

AAC TGC TAC AGG GAC GCG ATT GTC ACC ACC TCC ATC AAC TCC CTG ACG 1169 Asn Cys Tyr Arg Asp Ala lie Val Thr Thr Ser lie Asn Ser Leu Thr 345 350 355

AGC TTC TCC TCC GGC TTC GTC GTC TTC TCC TTC CTG GGG TAC ATG GCA 1217 Ser Phe Ser Ser Gly Phe Val Val Phe Ser Phe Leu Gly Tyr Met Ala 360 365 370

CAG AAG CAC AGT GTG CCC ATC GGG GAC GTG GCC AAG GAC GGG CCA GGG 1265 Gin Lys His Ser Val Pro lie Gly Asp Val Ala Lys Asp Gly Pro Gly 375 380 385

CTG ATC TTC ATC ATC TAC CCG GAA GCC ATC GCC ACG CTC CCT CTG TCC 1313 Leu lie Phe lie lie Tyr Pro Glu Ala lie Ala Thr Leu Pro Leu Ser 390 395 400

TCA GCC TGG GCC GTG GTC TTC TTC ATC ATG CTG CTC ACC CTG GGT ATC 1361 Ser Ala Trp Ala Val Val Phe Phe lie Met Leu Leu Thr Leu Gly lie 405 410 415 420 GAC AGC GCC ATG GGT GGT ATG GAG TCA GTG ATC ACC GGG CTC ATC GAT 1409

Asp Ser Ala Met Gly Gly Met Glu Ser Val lie Thr Gly Leu lie Asp

425 430 435

GAG TTC CAG CTG CTG CAC AGA CAC CGT GAG CTC TTC ACG CTC TTC ATC 1457

Glu Phe Gin Leu Leu His Arg His Arg Glu Leu Phe Thr Leu Phe lie

440 445 450

GTC CTG GCG ACC TTC CTC CTG TCC CTG TTC TGC GTC ACC AAC GGT GGC 1505

Val Leu Ala Thr Phe Leu Leu Ser Leu Phe Cys Val Thr Asn Gly Gly

455 460 465

ATC TAC GTC TTC ACG CTC CTG GAC CAT TTT GCA GCC GGC ACG TCC ATC 1553 lie Tyr Val Phe Thr Leu Leu Asp His Phe Ala Ala Gly Thr Ser lie 470 475 480

CTC TTT GGA GTG CTC ATC GAA GCC ATC GGA GTG GCC TGG TTC TAT GGT 1601

Leu Phe Gly Val Leu lie Glu Ala lie Gly Val Ala Trp Phe Tyr Gly

485 490 495 500

GTT GGG CAG TTC AGC GAC GAC ATC CAG CAG ATG ACC GGG CAG CGG CCC 1649

Val Gly Gin Phe Ser Asp Asp lie Gin Gin Met Thr Gly Gin Arg Pro

505 510 515

AGC CTG TAC TGG CGG CTG TGC TGG AAG CTG GTC AGC CCC TGC TTT CTC 1697

Ser Leu Tyr Trp Arg Leu Cys Trp Lys Leu Val Ser Pro Cys Phe Leu

520 525 530

CTG TTC GTG GTC GTG GTC AGC ATT GTG ACC TTC AGA CCC CCC CAC TAC 1745

Leu Phe Val Val Val Val Ser lie Val Thr Phe Arg Pro Pro His Tyr

535 540 545

GGA GCC TAC ATC TTC CCC GAC TGG GCC AAC GCG CTG GGC TGG GTC ATC 1793

Gly Ala Tyr lie Phe Pro Asp Trp Ala Asn Ala Leu Gly Trp Val lie 550 555 560 GCC ACA TCC TCC ATG GCC ATG GTG CCC ATC TAT GCG GCC TAC AAG TTC 1841 Ala Thr Ser Ser Met Ala Met Val Pro lie Tyr Ala Ala Tyr Lys Phe 565 570 575 580

TGC AGC CTG CCT GGG TCC TTT CGA GAG AAA CTG GCC TAC GCC ATT GCA 1889 Cys Ser Leu Pro Gly Ser Phe Arg Glu Lys Leu Ala Tyr Ala lie Ala 585 590 595

CCC GAG AAG GAC CGT GAG CTG GTG GAC AGA GGG GAG GTG CGC CAG TTC 1937 Pro Glu Lys Asp Arg Glu Leu Val Asp Arg Gly Glu Val Arg Gin Phe 600 605 610

ACG CTC CGC CAC TGG CTC AAG GTG TAGAGGGAGC AGAGACGAAG ACCCCAGGAA 1991 Thr Leu Arg His Trp Leu Lys Val 615 620

GTCATCCTGC AATGGGAGAG ACACGAACAA ACCAAGGAAA TCTAAGTTTC GAGAGAAAGG 2051

AGGGCAACTT CTACTCTTCA ACCTCTACTG AAAACACAAA CAACAAAGCA GAAGACTCCT 2111

CTCTTCTGAC TGTTTACACC TTTCCGTGCC GGGAGCGCAC CTCGCCGTGT CTTGTGTTGC 2171

TGTAATAACG ACGTAGATCT GTGCAGCGAG GTCCACCCCG TTGTTGTCCC TGCAGGGCAG 2231

AAAAACGTCT AACTTCATGC TGTCTGTGTG AGGCTCCCTC CCTCCCTGCT CCCTGCTCCC 2291

GGCTCTGAGG CTGCCCCAGG GGCACTGTGT TCTCAGGCGG GGATCACGAT CCTTGTAGAC 2351

GCACCTGCTG AGAATCCCCG TGCTCACAGT AGCTTCCTAG ACCATTTACT TTGCCCATAT 2411

TAAAAAGCCA AGTGTCCTGC TTGGTTTAGC TGTGCAGAAG GTGAAATGGA GGAAACCACA 2471

AATTCATGCA AAGTCCTTTC CCGATGCGTG GCTCCCAGCA GAGGCCGTAA ATTGAGCGTT 2531

CAGTTGACAC ATTGCACACA CAGTCTGTTC AGAGGCATTG GAGGATGGGG GTCCTGGTAT 2591

GTCTCACCAG GAAATTCTGT TTATGTTCTT GCAGCAGAGA GAAATAAAAC TCCTTGAAAC 2651 CAGCTCAGGC TACTGCCACT CAGGCAGCCT GTGGGTCCTT GTGGTGTAGG GAACGGCCTG 2711

AGAGGAGCGT GTCCTATCCC CGGACGCATG CAGGGCCCCC ACAGGAGCGT GTCCTATCCC 2771

CGGACGCATG CAGGGCCCCC ACAGGAGCAT GTCCTATCCC TGGACGCATG CAGGGCCCCC 2831

ACAGGAGCGT GTACTACCCC AGAACGCATG CAGGGCCCCC ACAGGAGCGT GTACTACCCC 2891

AGGACGCATG CAGGGCCCCC ACTGGAGCGT GTACTACCCC AGGACGCATG CAGGGCCCCC 2951

ACAGGAGCGT GTCCTATCCC CGGACCGGAC GCATGCAGGG CCCCCACAGG AGCGTGTACT 3011

ACCCCAGGAC GCATGCAGGG CCCCCACAGG AGCGTGTACT ACCCCAGGAT GCATGCAGGG 3071

CCCCCACAGG AGCGTGTACT ACCCCAGGAC GCATGCAGGG CCCCCATGCA GGCAGCCTGC 3131

AGACCAACAC TCTGCCTGGC CTTGAGCCGT GACCTCCAGG AAGGGACCCC ACTGGAATTT 3191

TATTTCTCTC AGGTGCGTGC CACATCAATA ACAACAGTTT TTATGTTTGC GAATGGCTTT 3251

TTAAAATCAT ATTTACCTGT GAATCAAAAC AAATTCAAGA ATGCAGTATC CGCGAGCCTG 3311

CTTGCTGATA TTGCAGTTTT TGTTTACAAG AATAATTAGC AATACTGAGT GAAGGATGTT 3371

GGCCAAAAGC TGCTTTCCAT GGCACACTGC CCTCTGCCAC TGACAGGAAA GTGGATGCCA 3431

TAGTTTGAAT TCATGCCTCA AGTCGGTGGG CCTGCCTACG TGCTGCCCGA GGGCAGGGGC 3491

CGTGCAGGGC CAGTCATGGC TGTCCCCTGC AAGTGGACGT GGGCTCCAGG GACTGGAGTG 3551

TAATGCTCGG TGGGAGCCGT CAGCCTGTGA ACTGCCAGGC AGCTGCAGTT AGCACAGAGG 3611

ATGGCTTCCC CATTGCCTTC TGGGGAGGGA CACAGAGGAC GGCTTCCCCA TCGCCTTCTG 3671

GCCGCTGCAG TCAGCACAGA GAGCGGCTTC CCCATTGCCT TCTGGGGAGG GACACAGAGG 3731

ACAGTTTCCC CATCGCCTTC TGGTTGTTGA AGACAGCACA GAGAGCGGCT TCCCCATCGC 3791 CTTCTGGGGA GGGGCTCCGT GTAGCAACCC AGGTGTTGTC CGTGTCTGTT GACCAATCTC 3851

TATTCAGCAT CGTGTGGGTC CCTAAGCACA ATAAAAGACA TCCACAATGG AAAAAAAAAA 3911

AGGAATTC 3919

(2) INFORMATION FOR SEQ ID NO:2 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 620 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID Nθ:2:

Met Ser Lys Ser Lys Cys Ser Val Gly Leu Met Ser Ser Val Val Ala 1 5 10 15

Pro Ala Lys Glu Pro Asn Ala Val Gly Pro Lys Glu Val Glu Leu lie 20 25 30

Leu Val Lys Glu Gin Asn Gly Val Gin Leu Thr Ser Ser Thr Leu Thr 35 40 45

Asn Pro Arg Gin Ser Pro Val Glu Ala Gin Asp Arg Glu Thr Trp Gly 50 55 60

Lys Lys lie Asp Phe Leu Leu Ser Val lie Gly Phe Ala Val Asp Leu 65 70 75 80

Ala Asn Val Trp Arg Phe Pro Tyr Leu Cys Tyr Lys Asn Gly Gly Gly 85 90 95

Ala Phe Leu Val Pro Tyr Leu Leu Phe Met Val lie Ala Gly Met Pro 100 105 110 Leu Phe Tyr Met Glu Leu Ala Leu Gly Gin Phe Asn Arg Glu Gly Ala 115 120 125

Ala Gly Val Trp Lys lie Cys Pro lie Leu Lys Gly Val Gly Phe Thr 130 135 140

Val lie Leu lie Ser Leu Tyr Val Gly Phe Phe Tyr Asn Val lie lie 145 150 155 160

Ala Trp Ala Leu His Tyr Leu Phe Ser Ser Phe Thr Thr Glu Leu Pro 165 170 175

Trp lie His Cys Asn Asn Ser Trp Asn Ser Pro Asn Cys Ser Asp Ala 180 185 190

His Pro Gly Asp Ser Ser Gly Asp Ser Ser Gly Leu Asn Asp Thr Phe 195 200 205

Gly Thr Thr Pro Ala Ala Glu Tyr Phe Glu Arg Gly Val Leu His Leu 210 215 220

His Gin Ser His Gly lie Asp Asp Leu Gly Pro Pro Arg Trp Gin Leu 225 230 235 240

Thr Ala Cys Leu Val Leu Val lie Val Leu Leu Tyr Phe Ser Leu Trp 245 250 255

Lys Gly Val Lys Thr Ser Gly Lys Val Val Trp lie Thr Ala Thr Met 260 265 270

Pro Tyr Val Val Leu Thr Ala Leu Leu Leu Arg Gly Val Thr Leu Pro 275 280 285

Gly Ala lie Asp Gly lie Arg Ala Tyr Leu Ser Val Asp Phe Tyr Arg 290 295 300

Leu Cys Glu Ala Ser Val Trp lie Asp Ala Ala Thr Gin Val Cys Phe 305 310 315 320 Ser Leu Gly Val Gly Phe Gly Val Leu lie Ala Phe Ser Ser Tyr Asn 325 330 335

Lys Phe Thr Asn Asn Cys Tyr Arg Asp Ala lie Val Thr Thr Ser lie 340 345 350

Asn Ser Leu Thr Ser Phe Ser Ser Gly Phe Val Val Phe Ser Phe Leu 355 360 365

Gly Tyr Met Ala Gin Lys His Ser Val Pro lie Gly Asp Val Ala Lys 370 375 380

Asp Gly Pro Gly Leu lie Phe lie lie Tyr Pro Glu Ala lie Ala Thr 385 390 395 400

Leu Pro Leu Ser Ser Ala Trp Ala Val Val Phe Phe lie Met Leu Leu 405 410 415

Thr Leu Gly He Asp Ser Ala Met Gly Gly Met Glu Ser Val He Thr 420 425 430

Gly Leu He Asp Glu Phe Gin Leu, Leu His Arg His Arg Glu Leu Phe 435 440 445

Thr Leu Phe He Val Leu Ala Thr Phe Leu Leu Ser Leu Phe Cys Val 450 455 460

Thr Asn Gly Gly He Tyr Val Phe Thr Leu Leu Asp His Phe Ala Ala 465 470 475 480

Gly Thr Ser He Leu Phe Gly Val Leu He Glu Ala He Gly Val Ala 485 490 495

Trp Phe Tyr Gly Val Gly Gin Phe Ser Asp Asp He Gin Gin Met Thr 500 505 510

Gly Gin Arg Pro Ser Leu Tyr Trp Arg Leu Cys Trp Lys Leu Val Ser 515 520 525 Pro Cys Phe Leu Leu Phe Val Val Val Val Ser He Val Thr Phe Arg 530 535 540

Pro Pro His Tyr Gly Ala Tyr He Phe Pro Asp Trp Ala Asn Ala Leu 545 550 555 560

Gly Trp Val He Ala Thr Ser Ser Met Ala Met Val Pro He Tyr Ala 565 570 575

Ala Tyr Lys Phe Cys Ser Leu Pro Gly Ser Phe Arg Glu Lys Leu Ala 580 585 590

Tyr Ala He Ala Pro Glu Lys Asp Arg Glu Leu Val Asp Arg Gly Glu 595 600 605

Val Arg Gin Phe Thr Leu Arg His Trp Leu Lys Val 610 615 620

(2) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 40 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

(iii) HYPOTHETICAL: YES

(iv) ANTI-SENSE: NO

(ix) FEATURE:

(A) NAME/KEY: -

(B) LOCATION: 1..40

(D) OTHER INFORMATION: /label= consensus

/note= "consensus sequence of VNTR element in 3' untranslated region of HUDAT cDNA" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3 :

AGGAGCGTGT ACTATCCCAG GACGCATGCA GGGCCCCCAC 40

(2) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 23 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (synthetic)

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: NO

(ix) FEATURE:

(A) NAME/KEY: -

(B) LOCATION: 1..23

(D) OTHER INFORMATION: /label= oligonucleotide

/note= "synthetic oligonucleotide T3-5LONG, upstream primer for PCR analysis of VNTR region of in 3' untranslated region of HUDAT gene "

(xi) SEQUENCE DESCRIPTION: SEQ ID Nθ:4:

TGTGGTGTAG GGAACGGCCT GAG 23

(2) INFORMATION FOR SEQ ID NO:5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (synthetic)

(iii) HYPOTHETICAL: NO

(iv) ANTI-SENSE: YES

(ix) FEATURE:

(A) NAME/KEY: -

(B) LOCATION: 1..24

(D) OTHER INFORMATION: /label= oligonucleotide

/note= "synthetic oligonucleotide, T7-3aLONG; downstream primer for PCR analysis of VNTR region of 3' untranslated region of HUDAT gene"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5

CTTCCTGGAG GTCACGGCTC AAGG 24

Claims (24)

What is claimed is :

1. An isolated, purified protein comprising the amino acid sequence described for Hdat in Figure 3 of the specification (seq. I.D. NO. 2) , and variants or portions thereof which retain the activity of said protein, said activity comprising the selective binding of dopamine, cocaine or functional analogs thereof.

2. An isolated, purified protein as recited in claim 1, wherein said biochemical activity further comprises transport of dopamine, cocaine or functional analogs thereof from one side of a membrane to the other.

3. An isolated cDNA comprising a nucleotide sequence which encodes the protein of claim 1.

4. An isolated cDNA comprising a nucleotide sequence which encodes the protein of claim 2.

5. An isolated cDNA comprising a nucleotide sequence as set forth in Figure 1 (SEQ. ID. NO. 1) of the Specification.

6. A plasmid DNA comprising the cDNA of claim 3, and a DNA sequence that provides for replication in a prokaryotic host cell and DNA sequences that provide for transcription of the cDNA in vitro, such that the resulting mRNA can be isolated and translated upon introduction into a eukaryotic cell type.

7. A plasmid DNA comprising the cDNA of claim 4, and a DNA sequence that provides for replication in a prokaryotic host cell and DNA sequences that provide for transcription of the cDNA in vitro, such that the resulting mRNA can be isolated and translated upon introduction into a eukaryotic cell type.

8. A eukaryotic cell line derived from a cell type that does not normally express dopamine transport activity at its surface that has been made to transport dopamine or to bind CFT by the introduction of the DNA of claim 2 into its genome.

9. A eukaryotic cell line derived from a cell type that does not normally express dopamine transport activity at its surface that has been made to transport dopamine or to bind CFT by the introduction of the DNA of claim 4 into its genome.

10. The cell line of claim 9 wherein the cell type is COS cells.

11. A method for screening cocaine antagonists which comprises measuring the inhibition of binding of cocaine, or of functionally equivalent cocaine analogs, to the surface of cells or to membrane preparations obtained from said cells, wherein said cells express the cDNA of claim 3.

12. A method for screening cocaine antagonists which comprises measuring the inhibition of cell toxicity due to MPP⁺ import into cells expressing the protein of claim 2 or transport across reconstituted membrane preparations obtained from such cells.

13. A method for screening therapeutic agents effective in the prevention or treatment of Parkinson's disease which comprises measuring the inhibition of cell toxicity due to MPP⁺ import into cells expressing the protein of claim 2, or measuring MPP⁺ transport across reconstituted membrane preparations obtained such cells.

14. A method for screening therapeutic agents effective in the prevention or treatment of Parkinson's disease which comprises measuring the inhibition of binding of cocaine, or of functioanlly equivalent cocaine analogs, to the surface of cells or to membrane preparations obtained from said cells, wherein said cells express the cDNA of claim 3.

15. A method for the screening of therapeutic agents effective in the prevention or treatment of diseases caused by abnormal dopamine transport which comprises measuring either the inhibition or facilitation of binding of cocaine, or of functionally equivalent cocaine analogs, to the cell surface of cells of the line or to membrane preparations obtained from the cell line of claim 9, by candidate compounds.

16. A method for the screening of therapeutic agents effective in the prevention or treatment of diseases caused by abnormal dopamine transport which comprises measuring the cell toxicity due to MPP⁺ import into cells of the line or transport across reconstituted membrane preparations obtained from the cell line of claim 9, by candidate compounds.

17. A method for the treatment of Parkinson's disease, Tourette's syndrome or other disease caused by abnormal HUDAT expression by a gene therapy technique comprising transplanting into a patient somatic cells which have been transformed with a recombinant DNA construction expressing the cDNA of claim 4, thus obtaining expression of said cDNA in said patient .

18. A method for diagnosing genetic variants in dopamine transporter protein utilizing a Southern blot based testing method which detects genetic rearrangements or deletions in the DAT gene, which comprises using the cDNA of claim 3 as a probe.

19. A method for diagnosing genetic variants in dopamine transporter protein as in claim 18, wherein the cDNA probe comprises DNA having the nucleotide sequence of Figure 1 (SEQ. ID. NO. 1) or a portion thereof.

20. A method for screening an individual for a genetic marker associated with substance abuse behavior which comprises analyzing the alleles of HUDAT present in the genotype of said individual.

21. The method of claim 20, wherein said alleles are variable number tandem repeat alleles.

22. A method for diagnosing variant expression of dopamine transporter utilizing a testing method based on the polymerase chain reaction which comprises using polynucleotide primers derived from the cDNA of claim 3.

23. A method for diagnosing variant dopamine transporter expression as in claim 22, wherein the polynucleotide primers are derived from the nucleotide sequence shown in Figure 1 (SEQ. ID. NO. 1) .

24. A method for detecting changes in the rate of release of human dopamine transporter from dopaminergic cells into cerebrospinal fluid that comprises measurement of peptides of the human dopamine transporter released into cerebrospinal fluid.