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WO1993008262A1 - Clone d'adn complementaire codant un transporteur exprimable de dopamine - Google Patents

Clone d'adn complementaire codant un transporteur exprimable de dopamine Download PDF

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WO1993008262A1
WO1993008262A1 PCT/US1992/008874 US9208874W WO9308262A1 WO 1993008262 A1 WO1993008262 A1 WO 1993008262A1 US 9208874 W US9208874 W US 9208874W WO 9308262 A1 WO9308262 A1 WO 9308262A1
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dopamine
pdat
cell
cdna
cells
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Susan G. Amara
John E. Kilty
Dominique Lorang
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Oregon Health and Science University
Oregon State
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Oregon Health and Science University
Oregon State
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes

Definitions

  • This invention pertains to the cloning of a. DNA sequence.
  • this invention pertains to the cloning of a complete complementary DNA (cDNA) sequence for a neurotransmitter transporter protein and the cloning of a portion of said cDNA sequence usable for high-stringency screening of such genes in animal cells.
  • cDNA complementary DNA
  • Transmission of a nerve impulse across a chemical synapse involves the secretion of neurotransmitter substances by the presynaptic neuron into the synaptic cleft. This facilitates the transmission of a chemical signal across the synaptic cleft to the postsynaptic neuron. Transmission of the chemical signal is normally transient. Otherwise, if the neurotransmitter substances persisted in the synaptic cleft, a new signal would not get through. Nervous tissue normally disposes of soluble or unbound neurotransmitter in the synaptic cleft by various mechanisms, including diffusion and enzymatic degradation. In addition, at most synapses, chemical signaling is terminated by a rapid reaccu ulation of neurotransmitter into presynaptic terminals.
  • This reaccumulation is the result of reuptake of the neurotransmitter by the presynaptic neuron.
  • reuptake of the neurotransmitter from the synaptic cleft is probably the most common mechanism used for terminating the chemical signal.
  • the various molecular apparatuses for reuptake are highly specific for such neurotransmitters as choline and the biogenic amines (low molecular-weight neurotransmitter substances such as dopamine, norepinephrine, epinephrine, serotonin, and histamine) .
  • These apparatuses are termed "transporters” because they transport the corresponding neurotransmitter from the synaptic cleft back across the cell membrane of the presynaptic neuron into the cytoplasm of the presynaptic terminus.
  • Certain psychotropic drugs such as cocaine and imipramine are effective because they block these reuptake processes by, for example, interfering with action of one or more transporters.
  • the administration of such drugs to block reuptake prolongs and enhances the action of neurotransmitters such as the biogenic amines.
  • These drugs also include therapeutic antidepressants and amphetamines.
  • dopamine transporters are structurally or functionally distinct from dopamine transporters in other dopaminergic systems such as the nigro-striatal pathway, the retina, the hypothalamus, or the olfactory bulb is an important step in understanding the neurobiologic basis of cocaine abuse. Also, elucidating the structural and functional distinctions between different types of transporters, including non- dopaminergic transporters, is important in understanding the cellular and molecular bases of behavior.
  • cDNA complementary DNA
  • pDAT a clone named "pDAT”.
  • KW27 another clone is provided, named "KW27”, which comprises a ca. 700 base-pair (bp) portion of the dopamine transporter cDNA.
  • bp base-pair
  • oligonucleotides were synthesized corresponding to regions of high sequence identity between the norepinephrine transporter (NET) and the ⁇ -aminobutyric acid transporter (GABAT) .
  • the oligonucleotides were used in polymerase chain reactions (PCR) with mRNA from mid-brain.
  • PCR polymerase chain reactions
  • the amplified DNA sequences were cut with a restriction endonuclease, electrophoresed, precipitated, and ligated into expression vectors to create a library.
  • KW27 One clone, designated "KW27", from the library had a 700-bp nucleotide sequence that was similar but not identical to a corresponding region in the NET gene sequence.
  • KW27 when radiolabeled and used as a probe in Northern blots of RNA isolated from rat brain tissues, hybridized specifically to a 3.6 kilobase (kb) mRNA isolated from the substantia nigra, a brain region known to be especially rich in dopaminergic neurons.
  • Radiolabeled KW27 was used to probe cDNA libraries from idbrain and substantia nigra at high-stringency. Selected "KW27-positive" clones having DAT-specific sequences at least 1.5 kb long were sequenced. One clone, named “pDAT”, had a complete open reading frame encoding a putative protein having DAT characteristics and exhibiting substantial amino-acid conservation with NET and GABAT.
  • the DAT-gene specificity of KW27 was demonstrated by in situ hybridization to sections of brain tissue. KW27 hybridized intensely and selectively to the substantia nigra, ventral tegmental areas, and the periphery of the olfactory bulb, all of which are known to be rich in dopaminergic neurons.
  • pDAT was transfected interspecifically into HeLa (non-neuronal) cells that normally do not accumulate dopamine, the cells acquired the ability to accumulate the neurotransmitter with accumulation kinetics that agreed substantially with the kinetics of dopamine accumulation by striatal synaptosomes.
  • FIG. 1 shows the complete amino sequences of the norepinephrine and GABA transporters as disclosed in the prior art, and the putative dopamine transporter protein encoded by pDAT dopamine transporter cDNA according to the present invention.
  • FIG. 2 shows the complete nucleotide sequences of pDAT and KW27.
  • FIG. 3 shows cumulative dopamine uptake by pDAT- transfected HeLa cells; the inset is an Eadie-Hofstee plot of the uptake.
  • FIG. 4 shows the effect of desipramine, cocaine, and azindol on dopamine uptake by pDAT-transfected HeLa cells .
  • a dopamine-transporter cDNA (DNA sequence complementary to a messenger RNA (mRNA) produced by the dopamine transporter gene) according to the present invention was isolated and cloned using a strategy that exploited certain similarities in the amino-acid sequences of other transporter proteins, namely the norepinephrine transporter (NET, responsible for uptake of norepinephrine, one of the "biogenic amine” group of neurotransmitters) and the ⁇ -aminobutyric acid transporter (GABAT, responsible for uptake of ⁇ - aminobutyric acid, one of the "amino acid” group of neurotransmitters) . It was hoped when we began these studies that the dopamine transporter (DAT) would exhibit a similar degree of amino-acid sequence conservation, which would provide a way to selectively isolate and clone cDNA encoding the DAT.
  • DAT dopamine transporter
  • the NET amino-acid sequence is shown in FIG. 1, as disclosed- in Pacholczyk et al., Nature 350:350 (1991) and U.S. Patent application Serial No. 07/676,980, filed on March 28, 1991, by the same authors as the Nature paper.
  • the amino-acid sequence of the GABAT is also shown in FIG. 1, as obtained from Guastella et al. , Science 249:1303- 1306 (1990) and Nelson et al. , FEBS Lett. 269:181-184
  • FIG. 1 the amino-acid sequences of NET and GABAT are shown in register to illustrate highly conserved regions (shaded areas) .
  • the brackets labeled with roman numerals designate regions believed to be transmembrane domains.
  • oligonucleotide primers to be used in a polymerase chain reaction (PCR) protocol to selectively isolate DAT cDNA from cells known to exhibit a high degree of DAT expression.
  • PCR polymerase chain reaction
  • KNGGGAFLIPY This first conserved region is referred to herein as the "KNG” region.
  • the second conserved region that was selected resides near the beginning of the transmembrane region designated “VI” and comprises the amino acids WIDAATQIFF. This second conserved region is referred to herein as the "WID” region.
  • WID This second conserved region.
  • FIG. l abbreviations for the amino acids are as indicated below:
  • amino-acid sequences of the KNG and WID regions enabled oligonucleotide primers for PCR to be designed comprising codons that would encode either the KNG or
  • the WID pool comprised the PCR "antisense" primers each having the following coding sequence:
  • the oligonucleotides comprising the KNG and WID pools were prepared using a conventional automated nucleotide synthesizer apparatus.
  • the first-strand DNA was used as a template in PCR reactions using the KNG and WID primer pools.
  • PCR reactions were performed using conventional techniques, Mullis and Faloona, Methods in Enzymol. 155:335 (1987), and the thermostable DNA polymerase from Thermus aquaticus. Saiki, et al., Science 239:487 (1988) .
  • Each PCR reaction contained cDNA synthesized from 50 ng of RNA.
  • Thermal cycling was carried out for 25 cycles, wherein each cycle comprised a regimen of 94°C for one min. , 47°C for 2 min. , and 72°C for 3 min. , followed by a soak at 72°C for 12 min.
  • the PCR reactions produced amplified DNA sequences presumably including a number of transporter sequences and, it was hoped, DAT sequences.
  • the amplified DNA sequences were cleaved using
  • Bluescript vectors are available from Stratagene, La Jolla, California. They contain the T7 promoter and are popular expression vectors.
  • the recombinant vectors were used to create a library of bacterial clones each containing a length of PCR-amplified DNA.
  • KW27 Upon sequencing the remaining clones, we found that one, which we designated "KW27", had a sequence about 700 base pairs (bp) long that was relatively similar to a corresponding region in the NET gene but less similar to a corresponding region in the GABAT gene. This preliminary result was in accordance with other research data indicating that DAT may be more similar to NET than to GABAT. Therefore, we strongly suspected that KW27 contained a DAT-specific sequence. If so, then the ca. 700-bp length of KW27 would be highly discriminating for the DAT gene if a DAT-specific probe could be made from KW27.
  • RNA isolated from various brain tissues was separated by size on denaturing agarose electrophoretic gels and transferred to nylon membranes according to the conventional "Northern" hybridization blotting technique. See, Alwine et al., Proc. Natl. Acad. Sci. USA 74:5350 (1977); Alwine et al. , Methods in Enzvmol. £8 . :220 (1979). The membranes were incubated with the radiolabeled probes. Hybridization of the ca. 700 bp KW27 fragment was very stringent. Subsequent autoradiography revealed that a 4-kilobase (kb) mRNA band originally isolated from the substantia nigra was strongly labeled with the KW27 probe.
  • kb 4-kilobase
  • KW27 enabled us to determine that there is a single dopamine transporter gene product encoded by the ca. 4- kb mRNA. The same mRNA appears to be expressed in all dopaminergic cell bodies including those of the nigro- striatal and meso-limbic systems.
  • radiolabeled KW27 was used to perform high-stringency screening of midbrain and substantia nigra cDNA libraries in an effort to find a full-length cDNA encoding DAT.
  • first-strand cDNA was generated from poly(A)-selected mRNA (Chirgwin et al., Bioche . 18:5294 (1979) ) isolated from substantia nigra and midbrain using RAV reverse transcriptase (Amersham Corp. , Arlington Heights, Illinois) and random hexanucleotide primers. The first-strand DNA was then used to produce double-stranded cDNA by conventional methods. cDNA having blunt ends was ligated to semi-Xho adaptors
  • Colony lifts (replica plates) of the resulting libraries were screened at high stringency using the radiolabeled KW27 probe.
  • “high stringency” means that an unusually high “melting” temperature was required to denature the hybrids, indicating that the hybrids comprised at least very nearly perfectly homologous sequences.
  • cDNAs from selected "KW27-positive” colonies were completely sequenced in both strands with Sequenase (U.S. Biochemical Corp., Cleveland, Ohio) using a set of overlapping exonuclease Ill-digested unidirectional deletions.
  • pDAT A clone, designated "pDAT", containing the complete DAT coding region was generated by ligating two overlapping clones at a Pfl MI site in the cDNAs.
  • pDAT comprised a single open reading frame encoding a putative protein having an amino-acid sequence and other characteristics strongly suggestive that it was a dopamine transporter.
  • the complete nucleotide sequence of pDAT is shown in FIG. 2. (and in Seq. ID No:l).
  • the probable amino-acid sequence of the DAT is also shown, as predicted by deciphering the nucleotide sequence of pDAT. Nucleotides are counted starting from the first residue in the insert. Stars are placed above every twentieth nucleotide, and every hundredth nucleotide is numbered. Numbering of amino-acid residues begins at the putative translational start site (ATG) with cumulative amino-acid counts shown in the right margin. Putative transmembrane domains are square-bracketed and underlined.
  • Potential glycosylation sites on a putative extracellular loop are denoted by double underlining (beneath four separate amino-acid triplets) .
  • Two potential sites for phosphorylation by protein kinase C in the N-terminal cytoplasmic domain and one potential site for phosphorylation by either protein kinase C or calmodulin-dependent protein kinase II .in the C-terminal cytoplasmic domain are indicated by solid triangles.
  • Abbreviations of the amino-acid residues are as tabulated above. Referring further to FIG.
  • the nucleotide sequence surrounding the upstream methionine (M) codon at nucleotide 90 has the best match to the Kozak consensus sequence, Kozak, Nucl. Acids Res. .15:8125 (1987), and therefore probably represents the translational start site.
  • a pyrimidine is present at the " third position upstream from the ATG. This has only been observed in three percent of mRNAs included in the Kozak study and may suggest that such mRNAs are less efficiently translated.
  • the PCR oligonucleotides used to isolate the KW27 probe hybridize to pDAT sequences indicated in FIG. 2 (and in Seq. ID No:l) by double underlining.
  • the sequence of the intervening nucleotides is identical to the sequence of the cloned PCR product, KW27. It will be appreciated by persons skilled in the art that the specific PCR oligonucleotides shown in FIG. 2 could have been any of the corresponding "sense” or “antisense” primers, as appropriate, encoding the "KNG” or "WID” amino acid sequences, respectively.
  • the N- and C- termini of DAT appear to be located cytoplasmically.
  • FIG. 1 there is a high degree of amino-acid sequence conservation between the putative transporter protein encoded by pDAT and the NET and GABAT proteins.
  • the pDAT protein and NET have about 64 percent amino-acid identity and the pDAT protein and GABAT have about 40 percent amino-acid identity.
  • the degree of similarity increases to 75 percent and 50 percent, respectively, if conservative amino-acid substitutions are made. Therefore, in accordance with data in the research literature pertaining to DAT function, the pDAT protein shares more similarity to the NET than the GABAT.
  • the amino-acid conservation between these three transporters is distributed fairly evenly throughout the amino-acid sequences thereof except for the cytoplasmic N-termini which are relatively transporter-specific. Even in the large extracellular loop between transmembrane domains III and IV, where our PCR analysis has identified transporter-specific sequences in other members of this transporter family, pDAT has 57 percent and 33 percent amino-acid identity to the NET and GABAT, respectively.
  • a cloned expressible dopamine transporter cDNA is provided for the first time.
  • pDAT can be readily incorporated into vectors and transfected into other cells, including non- neuronal cells and cells from different animal species. Such pDAT-transformed cells produce functional dopamine transporter that appears to integrate functionally into the cell membrane.
  • the activity of pDAT-encoded dopamine transporter in such cells is sensitive to antagonists of transporter function, including cocaine.
  • the present invention also provides, for the first time, a cloned cDNA segment (KW27) capable of hybridizing to a portion of the DAT gene with high stringency.
  • KW27 makes possible, for the first time, the rapid screening of central nervous system tissues so as to isolate DAT gene sequences therefrom. Screening methodology would be as described in detail herein or other methodology familiar to skilled artisans. Since research data strongly indicate that similar transporters are highly conserved among different animal species, particularly mammalian species, it will be readily appreciated by persons skilled in the art that KW27 can be used to isolate dopamine-transporter gene sequences in a large variety of animal species.
  • 35 S-labeled sense and antisense cRNA transcripts of KW27 were synthesized using T7 and T3 RNA poly erases and used to probe cryostat sections of perfusion-fixed rat brain.
  • Anesthetized Sprague-Dawley rats were initially perfused transcardially with ice- cold 4% paraformaldehyde solution followed by a second solution of 4% paraformaldehyde and 0.05% glutaraldehyde in borate buffer at pH 9.5.
  • Twenty-micron cryostat sections mounted on slides were processed through a mild proteinase K treatment (i.e. 10 ⁇ g/mL at 37°C for 30 min.), followed by acetylation and dehydration steps.
  • Sections were hybridized with radiolabeled denatured KW27 probe (1.5xl ⁇ 7 dpm/mL in a hybridization buffer containing 50% formamide, 0.25 M NaCl, lx Denhardt's solution, and 10% dextran sulfate.) Hybridization time was about 20 hours at 60°C. Sections were subsequently washed in decreasing concentrations of SSC, digested with RNase A (20 ⁇ g/mL at 37°C for 30 min.) and washed at a final stringency of O.lx SSC (wherein "SSC” denotes standard sodium citrate solution as known in the art) at 65-75°C in O.l SSC.
  • SSC standard sodium citrate solution as known in the art
  • Example 2 To further confirm that pDAT represents the actual dopamine transporter and not a related gene product coincidentally expressed in dopaminergic neurons, pDAT was introduced into HeLa cells. HeLa cells, which are derived from a human cervical carcinoma, are non-neural cells widely used in the art. These cells are normally incapable of transporting dopamine.
  • pDAT was introduced into HeLa cells by an infection/transfection technique employing the T7 promoter present in the Bluescript SKII(-) vector and T7 polymerase encoded by a vaccinia virus vector as described by Blakely et al. , Anal. Biochem. 194:302 (1991); and Fuerst et al., Proc. Natl. Acad. Sci. USA 83.:8122 (1986).
  • HeLa cells were plated in DMEM (Dulbecco's Minimal Essential Medium) , 5% FBS (Fetal Bovine Serum) in 24-well plates at l-2xl0 5 cells per well and infected with a T7 RNA polymerase-encoding vaccinia virus at a multiplicity of infection of 10 pfu/cell (wherein “pfu” denotes plaque-forming units, a measure of virus "concentration”) .
  • DMEM Disbecco's Minimal Essential Medium
  • FBS Fetal Bovine Serum
  • FIG. 3 comprises plots of the kinetics of dopamine uptake into pDAT-transfected HeLa cells.
  • the larger plot shows a time-course of labeled dopamine accumulation in HeLa cells transfected with the pDAT- containing vector. As can be seen, transfected HeLa cells demonstrated saturable dopamine accumulation.
  • the inset plot is an Eadie-Hofstee plot of initial velocity data.
  • the Eadie-Hofstee plot enabled us to determine the value of K M , the Michaelis constant. of dopamine uptake by the dopamine transporter. In the Eadie- Hofstee plot, -K M is equal to the slope of the line.
  • Example 2 was performed similar to Example 2 in which pNET- and pDAT-transfected HeLa cells were incubated with 50 nM 3 H-dopamine.
  • the cells were also exposed to various concentrations of three well-characterized antagonists of catecholamine transport in synaptosome preparations: disipramine (a tricyclic antidepressant which is a norepinephrine transporter antagonist) , cocaine (a known antagonist of reuptake of biogenic amines such as dopamine, serotonin, and norepinephrine) , and mazindol (which blocks catecholamine transporters with high affinity) .
  • disipramine a tricyclic antidepressant which is a norepinephrine transporter antagonist
  • cocaine a known antagonist of reuptake of biogenic amines such as dopamine, serotonin, and norepinephrine
  • mazindol which blocks catecholamine transporters with high affinity
  • FIG. 4 shows representative dose-response curves for the inhibition of dopamine transport in pDAT- transfected HeLa cells by the three antagonists.
  • Parallel experiments (curves not shown) were performed wherein dopamine transport was determined in pNET- transfected HeLa cells in the presence of various doses of the three antagonists.
  • the ordinate of FIG. 4 is specific dopamine uptake expressed as a percent of dopamine transport by the pDAT-transfected cells in the absence of inhibitor. Each data point reflects the mean +/- one standard error of the mean. From these plots, inhibition constants (K r values) were determined by ascertaining the antagonist concentration that caused a fifty-percent level of specific dopamine uptake.
  • Inhibition constants of dopamine transport in pNET- transfected cells were 2 nM, 4 nM, and 200 nM for mazindol, desipramine, and cocaine, respectively. From the data of FIG. 4, inhibition constants of dopamine transport in pDAT-transfected cells were 70 nM, 2 ⁇ M, and 4 ⁇ M, for mazindol, cocaine, and desipramine, respectively. This rank K ⁇ order for pDAT-transfected cells agrees with the ranked order of potency that would be expected for the effect of mazindol, cocaine, and desipramine on dopamine transport.
  • pDAT encodes a protein exhibiting various properties of the native dopamine transporter, including an expected rank order of pharmacological inhibition by transporter antagonists.
  • a cDNA clone according to the present invention can be inserted into .
  • any of a number of expression vectors particularly in view of the fact that the entire nucleotide sequence of pDAT is disclosed.
  • suitable vectors will have at least a functional promoter.
  • the vector be expressible in the target cells of interest.
  • the availability of a DAT cDNA clone according to the present invention now allows pharmacologic studies of dopamine transport to be undertaken in transfected cells devoid of vesicular storage compartments and free from the obfuscating influences of other transport pathways.
  • pDAT permits high-level expression of the dopamine transporter to be attained in a variety of mammalian and other animal systems which heretofore has not been possible. It will also be appreciated that pDAT allows the development of far more sensitive assay systems for ascertaining the effects of various drugs on transport function than has heretofore been possible.
  • the pDAT clone can also be used in conjunction with various animal-cell expression systems (transfected by pDAT) to provide a screen for identifying more selective pharmacologic agents that affect dopamine transport function.
  • the pDAT clone can be used to ascertain potential side effects of various pharmacologic agents related to their ability to interact with the dopamine transporter.
  • pDAT can be used diagnostically for imaging studies involving such compounds and other potential agents.
  • the ability to assess the relative affinities of various agents for the transporter in pDAT-transfected cell lines can serve as a useful screen for other potentially neurotoxic agents and can suggest therapeutic options for acute intoxications.
  • previous studies have relied upon laboratory animals as sources of membranes with transport activities, the use of animal cell lines transfected with pDAT for this and other research offers substantial advantages.

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Abstract

ADN complémentaire cloné codant un transporteur fonctionnel et exprimable de dopamine, et partie d'environ 700 paires de bases dudit ADN complémentaire de dopamine, apte à servir de sonde spécifique de transporteurs de dopamine, constituée de polynucléotides provenant de diverses cellules animales. Les cellules non nerveuses transfectées à l'aide de l'ADN complémentaire cloné acquièrent une capacité d'absorption de dopamine. L'absorption par ces cellules transfectées peut être inhibée par divers médicaments inhibiteurs d'absorption présentant une inhibition relative d'absorption de dopamine qui correspond aux effets relatifs desdits médicaments sur les neurones dopaminergiques, ce qui confirme que ledit ADN complémentaire code une protéine de transporteur fonctionnel de dopamine. Le clone d'ADN complémentaire rend possibles des études bien contrôlées du transport de dopamine dans les systèmes cellulaires. La partie d'environ 700 paires de bases est adaptée notamment au dépistage très stringent de polynucléotides spécifiques de transporteurs de dopamine, dans les cellules et tissus provenant de diverses espèces animales.
PCT/US1992/008874 1991-10-25 1992-10-16 Clone d'adn complementaire codant un transporteur exprimable de dopamine Ceased WO1993008262A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993024628A3 (fr) * 1992-06-01 1994-02-03 Us Health SEQUENCE D'ADNc CODANT LE TRANSPORTEUR DE DOPAMINE HUMAIN
US5756307A (en) * 1991-09-20 1998-05-26 The United States Of America As Represented By The Department Of Health And Human Services Sequence of human dopamine transporter cDNA

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
JOURNAL OF NEUROCHEMISTRY, Vol. 54, No. 2, issued 1990, BANNON et al., "Expression of a Human Cocaine-Sensitive Dopamine Transporter in Xenopus Laevis Oocytes", pages 706-708. *
NATURE, Vol. 330, issued 26 November 1987, HEDIGER et al., "Expression Cloning and cDNA Sequencing of the Na+/Glucose Co-Transporter", pages 379-381. *
NATURE, Vol. 350, issued 28 March 1991, PACHOLCZYK et al., "Expression Cloning of a Cocaine- and Antidepressant-Sensitive Human Noradrenaline Transporter", pages 350-354. *
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES USA, Vol. 85, issued December 1988, BLAKELY et al., "Expression of Neurotransmitter Transport from Rat Brain mRNA in Xenopus Laevis Oocytes", pages 9846-9850. *
SCIENCE, Vol. 249, issued 14 September 1990, GUASTELLA et al., "Cloning and Expression of a Rat Brain GABA Transporter", pages 1303-1306. *
SCIENCE, Vol. 254, issued 25 October 1991, KILTY et al., "Cloning and Expression of a Cocaine-Sensitive Rat Dopamine Transporter", pages 578-579. *
SCIENCE, Vol. 254, issued 25 October 1991, SHIRAADA et al., "Cloning and Expression of a Cocaine-Sensitive Dopamine Transporter Complementary DNA", pages 576-578. *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5756307A (en) * 1991-09-20 1998-05-26 The United States Of America As Represented By The Department Of Health And Human Services Sequence of human dopamine transporter cDNA
WO1993024628A3 (fr) * 1992-06-01 1994-02-03 Us Health SEQUENCE D'ADNc CODANT LE TRANSPORTEUR DE DOPAMINE HUMAIN

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