Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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SEQUENCE OF HUMAN DOPAMINE TRANSPORTER cDNA
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.
The dopamine transporter that acts to take
released dopamine back up into presynaptic terminals
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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
l0 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
1S components (E. J. Devor and C.R. Cloninger, Annu. Rev.
Genet. 23,.19 (i989): D: Pawls and J. Leckman, New Eng.
J. Med: 315, 993 (1986); R. Dickens at al., Arch. Gen.
Psychiatry ~8, 19 (1991)): thus identification of
linkage markers for the human DAT is also of interest.
2'o 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.
2-5 Snyder, Eds. (Plenum, N~w 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
transpartera ~iD ~ fash~icn that , correlates well with
30 their behavioral reinforcing and psychomotor stimulant
properties; these transporters are thus the principal
brain "cocaine receptor8" related to drug abuse (M. C.
Ritz, R.J. Lamb, S.R. Goldberg, M.J. Kuhar, Science
23,1219 ('1987); J. 8ergman, B. K. Madras, S. E.
WO 93/24628 ,~ PCT/US93/05179
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Johnson, R. O. 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~ (1-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. taros et al. ,
FEBS Lett. 295, 149 (1992): J.E. Kilty et al., Science
254:, 578 (19~~,) : ; S.. ;~Sk~;imada et ,al. , Science 254, 576
(1991)': T.B. Usdin et al., Proc. Natl. Acad. Sci. USA
88, 11168 (1991) provides hybridization pxobes useful
for isolation of their human cognate.
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_Summarv of the Invention
Described herein is a cDNA (pcfiUDAT), which
encodes the human dopamine transporter protein (HUDAT).
Also described are unique features of the nucleotide
sequence of the pcHtIDAT 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 pcFiUDAT 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 (FiUDAT) 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,az~d/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.
VI~O 93/24628 . PCT/US93lOS179
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A third object of the invention is to provide
diagnostic means for assessing HUDAT expression in
patients by DNA- or antibody- based tests anc'~ for
assessing the onset or progression of disease by assay
5 of HUDAT degradation.
These and other objects are accomplished by
providing a cDNA encoding the dopamine transporter
protein and a gurified polypeptide conferring upon
cells the phenotype of dopamine uptake from the
l0 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 tra,nsporter~ , ,pxotein: 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
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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 48 shows the
same DNA, but hybridized with the Taq492 probe, which
corresponds to nucleotides 301-793 of the pcHUDAT
sequence.
~15 Due' AILSD. INSCRIPTION 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
~e .original amino acid sequence is modified or changed
v 20 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 HLTDAT are
25 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 engin~egri~g.., ,; , , : ; , , .
Expression of heterologous proteins in ~. coli is
30 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
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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 pGE~ 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 Ba_c~,illus strains.
Expression of proteins in bacteria is well-
reviewed in "Current Protocols in Molecular Biology",
which is published with quarterly updates by Wiley
Interscience.
WO 93/24628 . PCT/US93/05179 s,,:~.:
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Expression of "foreign" proteins in mammalian
cells can be accomplished in two general fashions.
Transient expression refers to the creation of a.~ool
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 repraducible
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 gromoters. 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
Pffectively 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.
Tk~e pre~~~red :embodiments ,of the ~,nvention 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
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considered to fall within the scope and spirit of the
instant application.
Example 1
Isolation and sgguencincr of cDNA encoding human
dopymine trar~orter
To isolate human cDNAs for the dopamine
transporter, cDNA libraries prepared from "substantia
nigra" and "brainstem" dissections containing cells
known to express the transporter were screened with
l0 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, Ia 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 pg/~1 in 2 x SSPE/0.1%SDS) to reduce
filter background, washed in 5 x SSC/0.5% SDS/imM 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 by Eco RI fragment of the rat dopamine transporter
cDNA6 (S. Shimada et al. , Science 254, 576 (1991) ) [~P]
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
sequences as described (S. Shimada et al. , Science 254,
576 (1991) ) . Sequence analysis was performed using the
*Trade-mark
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GCG software package (J. Devereaux, et al.., Nucleic
Acids Res. i2, 387 (1984)).
Screening of more than 2 x 106 plaques from the
substantia nigra library produced no positives.
5 Screening 1 x 106 plagues 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
10 (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 by 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
* Trade-mark
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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
DAT1 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 DAT1.
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 DAT1 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 xegiQns; 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
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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 far 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
glycosylatibn.
The repeated motifs in the 3' untranslated regions
of these cDNAs are another interesting difference from
the rat sequences 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, MendeTian Inheritance is Man, 9th edn. , Johns
Hopkins University Press, Baltimore, 1990, 2028 p.).
The rat sequence does demonstrate 25 copies of a small
WO 93124628 PCT/US93/05179
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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 genes 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.
~xam~le 2
:Restriction Fragment Length Polvmorohism IRFLPI
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 .~~nd ; [32P~]~dCTP or by random priming o~
these two hybridized oligonucleotides, as described (A.
Feinberg and B. Vogelstein, Analyt. Biochem. 132, 6-9
(1982): 5. Shimada et al., Science 254, 576-578
(191)). Identical results were also obtained using a
random-priming labeled 492 base pair cDNA fragment (Taq
,m.v.:..; _ . . ' ; .. . <, , ~.;:. , :; , ..
WO 93124628 PCl'/US93/05179 ; .: ,
14
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 radiolabeZed pHCDAT 7
after digestion with Alu I, Bam HI, Eco RI, Hae III,
Hind III, I~sp I, and Rsa I. Three other enzymes, Pst
z, Hinf I and Tag I revealed polymorphisms. We focused
on the polymorphisms identified by Taa I. When probed
with radiolabeled pHCDAT 7, more than six bands were
obtained from Taa 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 A1 and A2. Tag
I A1 and A2 RFLP frequencies are presented the Table.
Of 272:"chromosomes fxom: 136 individuals,examined 36%
showed the A1 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 A1 RFLP (xz=7.45, p< 0.01).
.~ ar': ~ ~ ., . .,., ,.. , .~..~. ~...... ...... ,.... .. . . ..;. _.... . '
~.: ~ . .",.,.. ~ ..,~. ....,; .. , ...,;.-:
WO 93/24628 PCT/US93/05179
t.. .
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
5 polymorghisms, because extreme variability of bands in
the initial Tact 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).
10 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
15 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
psycl~ostimulant abuse, Parkinsonism, and Tourette°s
syndrome (E. J. Devor and C.R. Cloninger, Annu. Rev.
Genet. 23 , 19-36 (1989): D. Pawls and J. Leckman, New
Eng. J. Med. 315, 993-997 (1986) : R. Dickens et al. ,
Arch. Gen. Psychiatry ~8, 19-28 (1991): M.C. Ritz et
al., Science 23?, 1219-122 3 (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 cD~l.As;and.RFLP information described here
should provide useful tools to study its possible role
in these and other human disorders.
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Example 3 Predictive)
A genetic component of substance abuse behavior
identified by RFLP analysis of tie human DAT ger~e~
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 2a,
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 ~3, 1131-1136 (1987); R.W: Pickens
et al:, Arch Gen Psychiatry 48, 19-28 (1991)).
2o 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. i, 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 ,strqngly towafid a possible direct
action for this highly-reinforcing drug in these
dopaminergic circuits (M.C. Ritz et al., Science 23~,
1219-1223 (1987); D.E. Grigoriadis et al., J Neurosci.
9, 2664-2670 (1989)).
WO 93/24628 PCT/US93/05179
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Blum, Noble and co-workers first reported that the
"Ai" T~gI restriction fragment length polymorphism
(RFLP) of the human dopamine D2 receptor gene (DRDZ,
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
l0 461 Caucasian "control" individuals are A1 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 associat~.ons between
RFLP and alcoholism, and provide evidence for a
ignificant 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. Polvdrug Abuse~ The Results of a~National~
Collaborative Study. New York, NY: Academic Press,
25'Tnc: 19?8): 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 ~ (~I,.;~t.: 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 (199I)):
WO 93/24628 ~ ~ ' " r PCT/US93/05179
t~ ;' ~'.:'
18
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' Tacrl A1 RFLP
examined in previous studies of alcoholics, and a more
5' TaaI 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 Ta~I A and B
markers in white and black individuals (Dr Bruce O'Hara
et al, wnpublished data). Substance. users were
identified according to two approaches. One group of
users met criteria for lifetime DSM-III-R jDiaq_nostic
and Statistical Manual of Mental Disorders, Revised
Third Edition. Washington, DC: American: Psychiatric
Association: 1987) psychoactive substance use
20~ 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 disglay significant heritability in males and
females (R. W. Pickens et al., Arch Gen Psychiatry ~t8,
19-28 (1991)). Control subjects were free of
significant lifetime substance use.
i), Subiect Recruitment:; : X88 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
WO 93/24628 . PCT/US93/05179
. fl n
19
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 Egid.
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. 9.7
users received both assessments. Written informed
consent was obtained from all subjects:
quantity-Fre~ency 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
l~.fetime Beak 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,, 1=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, alGOho,l,, marijuana, or nicotine ~c 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, andjor
up to moderate use of other drugs; "3",_ heavy use of
any illicit drug: Thus, neither heavy use of alcohol
WO 93/24628 PCf/US93J05179
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
5 scores of 2 or 3.
DSM-III-R Diactnoses: Trained interviewers
administered the Diagnostic Interview Schedule Version
III Revised (DIS-III-R, L.N. Robins et al.,NIMH
Diacxnostic Tnterview Schedule Version III Revised
10 (Version 11/7/89). Department of Psychiatry,
Washington University School of Medicine, St. Louis,
MO.) to provide lifetime DSM-ITT-R diagnoses of
psychoactive substance use disorders including nicotine
and alcohol.
15 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-ITI-R psychoactive substance
20 'use disorders by the DIS-III-R~ (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 nsychotic screen)
SCIL~~P (W/Psychotic Sereenl - 5/1/89). Biometrics
Research Department, New York State Psychiatric
Institute, New York, New York)(SLID; 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-assg,ssed sub~j~cts and, the 17 SCID-assessed
subjects, DUS ratings were completed without knowledge
of psychiatric assessment information. Genotypes were
not available for 17 sub j ects assessed with the SCID
and were not included in the genetic analyses.
WO 93/24628 PCT/US93/05179
21
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 = newer used alcohol
1 up to 4 drinks per drinking
occasion, fewer
than 10 drinking occasions/month
2 - up to 4 drinks per drinking
occasion, more
than 10 drinking occasions/month,
OR,
more than 4 drinks per drinking
occasion,
but fewer than 1~0 drinking
occasions/month
3 - 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/w~ek
Opiates 2 _ 2 to 6 times/week , spending $30 to
$100/day
3 - daily use, typically spending >
$loo/day
Cocaine 0 _ never used cocaine
1 _ less than 2 grams per week (up to
$150/we~k):
typical use - about 1 gram per month
2 - 2 to 4 grams per week (more than
$150/week
,
butless 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)
WO X3124628 PCTlUS93105179
~~a6~~~r~
2z
rlarijuana 0 = never used marijuana
1 = up to one joint/day
2 = 2 to 3 joints ger 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 b 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
CA 02136087 2003-06-18
23
ii) DNA Extraction and Anal~rsis: 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 ~g of this DNA was
digested with ~agI 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 NaPO' (pH 6.8), 1% SDS,
1mM EDTA, 2.5 x Denhardt's solution, 200 ~g/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 .1 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 ~gI A RFLP
status was determined, 3zP decay allowed re-
hybridization of the same blots with hybridization
probe for ~gI 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
* Trade-mark
WO 93/24628 ' ~ ~~ PCT/US93/OS179
i,:
z4
0.1~ SDS to remove residual hybridized probe. RFLP
status was assigned by two independent raters unaware
of the clinical status of the subjects. M
iii) Hybridization probes: A 1.7 kb BamHI fragment of
the human genomic clone encoding the dopamine DZ
receptor (.lhD2Gl) was subcloned into the ,~amHI site on
bluescript SK+ to produce phD2-9, which was used to
detect A1, 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). JlhD2G2 was~used to detect the TaQI "B"
patterns. DNAs were radiolabelled using random priming
and 32P-CTP to specific activities of approximately 109
cpm/~g (A. Feinberg and 8 Vogelstein, Anal Biochem.
137, 266-267 (I984)).
iv) 'nalyses:
a~ Association analyses: A two-tailed Pearson chi
square test (with Yates' correction for
continuity) was used to evaluate the association
between A1 RFLP presence and substance use/abuse;
the 'same analysis were repeated for the B2 RFLP.
Association was first tested contrasting controls
and substance users meeting criteria for any
lifetime DSM-III-R substance dependence disorder.
Next, controls,,wer~ 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 Qther data~ Pooled
~gI A1 RFLP data from ARC users was compared with
WO 93/24628 ~ ~ ~ ~ ~ j r' PCT/US93/05179
values obtained for Caucasian controls in
other studies.
_c) Subtractinct heavy alcohol users: DUS-assessed
users free of heavy alcohol use were compared with
5 controls to test whether the associations observed
might be attributed solely to alcohol.
TaQI A and B RFLPs were assigned with 100%
agreement between two independent raters.
Substance use assessment by means of the Drug Use
'l0 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 = U . 94 ) , while
test-retest reliability correlations for individual
15 drugs ranged, from 0.53 to 0.94 (median = 0.78) . For 35
subjects with DIS-III-R or SLID 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
20 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.
ac;I A and B RFLP frequencies for substance-using
25 and control subjects are presented in Table II. For
the Tacrl Bl RFLP, a significant association was found
comparing users with at least one lifetime DIS-III-R
Substance Use ., Di;sorder~ ;diagnos,is arid DUS-assessed ,
controls (XZ = 6.74, p < 0.01). For the ~gI A1 RFLP,
analysis of the same groups revealed a significant
association (x2 - 3.98, p < 0.05). Comparison of DUS-
assessed users to DUS-assessed controls revealed a
significant association for the TaQI B1 RFLP (x2 = 5.45,
WO 93/24628 PC 1'/US93/05179
>1~~~J~~~
26
p < 0.02) and a trend towards significant association
for the TaQI A1 RFLP (x2 = 3.14, p < 0.08). Table III
presents TaQI A and B genotypes (homoxygotes "'and
heterozygotes) for DUS-assessed controls and users.
;: , - ;, .;,,
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S'VO 93/24628 PCT/US93/05179
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WO 93124628 ~ ~ ~ ~ ~'~ ~ PCT/US93/OSi79
29
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~'O 93/24628 ~ . fCI"1US93/05179
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No significant differences in RFLP
frequencies were found between DUS-assessed
substance users and DIS-III-R-assessed users ( for
TaaI Al, X2 = 0.005, ns; for TaQI B1, x2 = 0.331,
5 ns). We thus reanalyzed the data by pooling
substance users assessed in both fashions for
comparison with controls. Analysis of TaaI B1
data (x2 = 6.31, p < 0.02) and T~crl A1 data (x2 =
4:46, p < 0.04) again revealed significant
10 associations.
Comparisons of TaQI A1 RFLP frequencies in
ARC users and controls from all other published
studies revealed significant associations when
the controls were assessed (XZ -. 15.41, p <
i5 O.OOZ), unassessed (xz 9:'31, p < 0.003), or
pooled (7C2 _ 14.80, g < 0.001)
To examine whether the effects noted could
be attributed chiefly to the extent of alcohol
intake, heavy alcohol users achieving DUS alcohol
20 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 TaaI~ B1 RFLP found in the user
gxoup . 3 2 . 3 % of al l polysubstance users and 3 2 . 9 %
25 of the 73 polysubstance users free of heavy
alcohol use displayed the ~agI ~Bl marker.
The hypothesis that individual differences
in substance abuse ;may: be due, in ;part, to
different dopamine D2 receptor alleles marked by
30 fag L 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
WO 93/2462$ ,..
PCT/US93/05179
31
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. ~0,
191-225 (1989)). In the current example,
significant associations With heavy substance use
or abuse were found consistently for the TacsI B1
RFLP and less consistently for the ~aqI A1 RFLP.
These findings provide preliminary evidence that
a more 5' Taal RFLP (B1) 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. ~olydrug Abuses The Results of a National
Collaborative Studv. 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
WO 93/24628 , - PCT/US93/05l'79
>~~~~~~'~-1
32
associations noted here, we might anticipate a
weaker association between- Ba 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 TaaI
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. Dickens ; et :al. , Arch Gen Psychiatry ~8, ;
19-28 (1991); C.R. Cloninger and T. Reich, In:
Kety SS, Rowland LP, Sidman RL, Matthysse SW,
eds. Genet3.cs of neurolociaal and ps~rchiatric
disorders. New York, NY: Raven Press: 1983; pp.
Wt~3 93/24628 ~ ~ ~ ~ d~ ~ ~~~ PCT/US93/05179
33
145-166). Reliability and validity of the
quantity-frequency approach to subj acts' drug use
were supgorted 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
SLID 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
(1.985) E N. Oskooilar et al. , DIS Newsletter 8,' 9-
10 (1991)).
Comparison of Tactl 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,
WO 93/24628 ~ PCT/US93/05179
34
however, that behavioral effects of a gene might
be differentially reflected in quantity/frequency
or in disease/disorder approaches to def fining the
affected group.
The RFLPs studied here are the result of
polymorphic Taal restriction sites in which °'A"
RFLPs are located ca. 9 kb 3' to the final axon
of the D2 receptor gene (O. Civelli, personal
communication) and "B" RFLPs are located near the
l0 first coding axon (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 TactI variations were maintained together
by linkage disequilibrium resulting in specific
haplotypes (G. R. Uhl et al., Arch Gen Psychiatry
20. 49, 157-160 (1992)): This linkage disequilibrium
does exist (X. Y. Hauge et al., Genomics 10, 527-
530 (1991): Dr. Bruce 0'Hara et a1, unpublished
data) . In our data, ~ for example, the expected
frequency of the A2/A2-B2/82 haplotype would be
43% based on the frequencies of the A2 and B2
allelic markers. However, the observed frequency
of this haplotype was 61% (x2 - 16.33, p <
0.0;001) (76% fpr 'c,~ntrols and ~57% for users, X~ _ ,
5.15, p < 0.03), indicating substantial linkage
disequilibrium.
The lack of strong association between DZ
receptor gene RFLPs and substance use evident in
W'O 93/24628 ~ ~ ~~ ~ ~ ~ ~'~ PGT/US93105179
this study is consistent with estimates of the
heritable components of alcoholism and drug abuse
(E. J. Devor arid C.R. Cloninger, Annu Rev Genet.
23, 19-36 (1989).; R.J. Cadoret et al., Arch Gen
5 Psychiatry 43, 1131-1136 (1987)). One recent
study of concordance rates for alcohalism 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. Dickens
10 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. ,
15 Genet Epidemiol. 4, 395-414 (1987); C.E. Aston
and S:Y. Hill, Am J Hum Genet. 46, 879-887
( 19!90) ) . 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
20 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.
25 To investigate the association between the
DAT gene and substance abuse behaviors, one can
make use of the variable number tandem repeat
(~Tg) at ~ the; 3' ~er~d ; o,f the., ,mRNA described in ,
example 1. Alternatively the Taql RFLP described
30 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
CA 02136087 2003-06-18
36
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, DNA is
obtained from leukocytes from research volunteers
as described above. Genomic DNA (40 ng) is
subjected to 35 cycles of amplification using
AmpliTaq*~NA 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-
Elmer). Oligonucleotides T3-SLONG (5'-
TGTGGTGTAGGGAACGGCCTGAG-3', SEQ. ID. NO. 4) and
T7-3aLONG (5'-CTTCCTGGAGGTCACGGCTCAAGG-3', SEQ.
ID. N0. 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 l0 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
* Trade-mark
11-'O 93124628 PCT/US93/05779
37
Blacks displayed 20% of the 9-copy variant, The
3' VNTR marker defined by 9 versus 10 copies csf
the 40 basepair repeat displayed no significant
linkage diseguilibrium with the more 5' TaqI RFLP
(X2 values were 5.51 and 4.62 for White and Black
subjects, respectively, with 8 degrees of
freedom, p > 0.2.)
Examble 4 (predictive)
Expression of HUDAT protein in Escherichia coli
and purification of the bacterially ext~ressed
protein
Any of several expression systems can be
utilized to obtain HUDAT protein expression in ~.
co i: 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 inse=t fragment encoding the HUDAT protein by
electrophoresis and elution from an agarose gel
by standard techniques. Oligonucleotides having
the: v 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 XbaI restriction
WO 93/24b28 PCT/US93/05179
:., ~v-~ ~~ ',
L~.~~~
38
site of the pFLAG vector (International
Biotechnologies, Inc.). The appropriate E. coli
host is transformed and colonies containing the
HLTDAT 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 enterokinase 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 asolated 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 puri:f ioation 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
WO 93/24628 ~ ~ ~ r; P~CT/US93/U5179
~13~~~~
39
DAT in the sample rather than labelled gamma-
amino butyric acid. The protocol is modified a~
required to allow the isolation of DAT as a
distinct protein by techniques known to a
practitioner of the art.
Examx~le 6 (predictive)
Diagnosis of deficiency, mutant or overexoression
of dobamine transporter by 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 pcI~JDAT. 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 'mutant's can 'be''dbserved~ 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
WO 93!24626 ,.. f PCT/US93/O5i 79 ~..
f..:.
alleles in a single experiment, which has been
described for the CFTR gene, mutants of which
cause cystic fibrosis (Erlich, H.A., et a1
Science 252:1643 (1991).
5 The FiUDAT 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
10 behavior or diseases associated with expression
of particular HUDAT alleles (See example 3).
Example.? (predictive
Use of dopamine transporter expression to
incorporate as Bart of overexpression of a panel
15 of dopaminerctic crepes to reconstruct a
dooamineraic cell fine for therany in human
diseases result~na from defective dopamine
transuorter expression.
cDNAs for' the human dopamine transporter,
0 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
2S growth in G418,r 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
WO 93/2Q628 ~~ '~ ~~ ~ .~'~ J ~/d PC'~'/US931OS179
41
integrate several of the plasmids. If the
plasmids cannot be introduced serially or
together in this direction, serial edition of
tyrosine hydroxylase and DOFA 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 HFLC analysis and radiochemical
l0 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 ~ cel'1 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.
.'~-'!'.: .'. '.: ..~-: .... : w ;..,..;.,'.., .."'.. ~.,,...,.;.'. ~. .....
.::'.~. ~. ....;.;,, i. ,.~~..: ~"'...~,'.. "....:.;. -;.;..., .,....".
,;...,.....,, ;..,.. .,..,."..,,...,
..~A,:",.,..,. , .. ,.,, _ ; ."... ".... ..,,~,: :.. ~..~. . .. .. ~. ",.,. .
...,._..~. ,: ~; .':~, :.:..~..."..._.,._. .' .,.,...,.; ,. , ; .1 .,.,:
WO 93/24628 ~ ~ PCT/iJS93/Oa179
~136~~~~
42
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 secLuences in HUDAT urotein
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 specifis amino acids in the
HUDAT protein. The ability of mutant
transporters to take up [3Fi] dopamine, [3H]
I~iPP+, and to bind' [3Fi] cocaine and cocaine
analogues (especially [3H] CFTj is tested as
described previously (S. Shimada et al., Science
254, X76 (1991)). The Amersham mutagenesis
2n system (version 2.1, technical bulletin code
RPN1523j can be used. Initial studies of mutants
of' the aspartic acid residue in txansmembrane
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
b~ind~ing. ' 'vThess ~r r'esvlts document ; that the ;
residues key to dopamine transport are not
identical to those crucial to cocaine binding:
thefirst transmembrane residue change of
aspartic acid (residue 79j to glycine .reduces
WO 93/24628 PCT/US93/OS179
~~1~~10.8~ ~ ~~
a , , ~~, ,~:
43
cocaine binding by 10%, but reduces dopamine
transport by over 95%. Mutations in the secozid
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.
ExamoZe'9 lpredictivel
alteration of carbohydrate structure in the
extracellu_~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 i's~'of' interest to inve$tigate 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-
PCT/US93/05179
1V0 93/24628 ~ ~ ~ ~ ~~~''1 ; . ~ . ....
U~~._ ~ ,, ~ ~~
44
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, compararison of the
wild--type and mutant protein, both untY~eated 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 propdrt'ies. 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
WO 93/24b28 ~ ~''_ ~? PCT/US93/05~79
~13~~'8~
functional studies are described by Shimada et
al . (S . Shimada et al . , Science 254, 576 ( 1991) )~~.
Exam»le 10 (predictive)
Gell lines expressing HtTDAT protein on the
5 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 upan the binding of dopamine
10 (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
15 candidate compound: Such assays are described
using cells expressing the rat DAT cDNA in
Shimada et al. (S. Sh~:mada et al., Science 254,
576 (1991) ) and can be applied as. well to cells
expressing the human DAT cDNA.
2p Example 11 ~~redictive~
~roductior of antibodies to ~IUDAT and use of same
os is test o do a 'ne 'c a 1 deat .
A. Production of polyclonal antibodies.
HLTDAT pro~ein,.obtained as.'. described above or
25 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
WO 93/24628 . ~ PCT/US93/85179
~..~
,;,: . :.
213~~'~~ ~~'~ ~ ~ : .
46
utilized directly or the antibody may be purified
from the serum by any commonly utilizdd
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:
Production of monoclonal antibodies to HUDAT
Monoclonal antibodies to HUDAT or to
particular epitopes of giUDAT 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, C.L. and
MacMahon-Pratt, D. J. Immuriol. 131, 1987-1993
( 1:983 ) )' : Alternatively, peripheral blood
l~aphocytes maybe 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 (198?)).
Cell lines producing anti-HUDAT antibodies are
' ' ider~ti~ied'~ ~ by ;' oontimdnly employed screening
;
techniques. Monoclonal antibody is then purified
by well known techniques from the supernatants of
large-scale cultures of the antibody producing
cells.
WO 93/24628 PCT/US93/05179
~136~8'~ ~ ~~ ~ ~~
47
C. Diagnosis of dopaminergic cell death. in yivo
by immunoassay of cerebrospinal fluid of ~'a
patient using anti-HUDAT antibodies.
The death of dopami.nergic 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 HTJDAT 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: Elwated bevels. 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.
WO 93/24628 PCT/US93/051.79
48
SEQUENCE LISTING
M
(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
(C) OPERATING SYSTEMa PC-DOS/MS-DOS '
(D) SOFTWARE: Patentln Release #1.0, Version #1.25
(vi).CURRENT APPLICATION DATA:
(A) APPLICATION NUMEER:
($) FILING DATE:
(C) CLASSIFICATION:
(viii) ATTORNEY~AGENT II~tF'ORM~LTION' ~ - , ~ '
(A) NAME: Murphy Jr., Gerald M.
(B) REGISTRATION NUMBER: 28,977
WO 93/24628 PCT/US93/05179
~13~~8~
49
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 703-241-1300 .~
(B) TELEFAX: 703-241-2848
(C) TELEX: 248345
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3919 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) IiYPOTHETICAL: NO
(v~) ORIGINAL SOURCE:
(A) ORGANISM: Homo sapiens
(F) TISSUE TYPE: brainstem
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 102..1961
(D) OTHER INFORMATTON: /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
;, j ~ t i :, ,
WO 93/2462$ PCT/US93/d5179
(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 Va1 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 Ile Leu Va1 Lys Glu
25 30 35
CAG AAC GGA GTG CAG CTC ACC AGC TCC ACC CTC ACC AAC CCG CGG CAG 257
Gln Asn G1y Va1 Gln Leu Thr Ser Ser Thr Leu Thr Asn Pro Arg Gln
40 45 50
AGC CCC GTGGAG GCCCAG GATCGGGAG ACCTGG GGCAAGAAG ATCGAC 305
Ser Pro ValGlu AlaGln AspArgGlu ThrTrp GlyLysLys IleAsp
55 60 S
6
TTT CTC CTGTCC GTCATT GGCTTTGCT GTGGAC CTGGCCAAC GTCTGG 353
Phe Leu LeuSer ValIle GlyPheAla ValAsp LeuAlaAsn ValTrp
70 75 90
CGG TTC CCCTAC CTGTGC TACAAAAAT GGTGGC GGTGCCTTC CTGGTC 401
Arg Phe ProTyr LeuCys TyrLysAsn GlyGly GlyAlaPhe LeuVal
g5 90 95 100
CCC TAC CTGCTC TTCATG GTCATTGCT GGGATG CCACTTTTC TACATG 449
Pro Tyr LeuLeu PheMet ValIleAla GlyMet ProLeuPhe TyrMet
,,. 105 , , ; 110, , 115
GAG CTG GCCCTC GGCCAG TTCAACAGG GAAGGG GCCGCTGGT GTCTGG 497
Glu Leu AlaLeu GlyGln PheAanArg G1uGly AlaAlaGly ValTrp
120 125 130
PCI'/US93/~5179
WO 93/~4b28
51
AAG ATC TGC CCC ATA CTG AAA GGT GTG GGC TTC ACG GTC ATC CTC ATC 545
Lys Ile Cys Pro I1e Leu Lys Gly Val Gly Phe Thr Val Ile Leu Ile
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 Ile Ile 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 Ile His Cys
165 170 175 180
AAC AACTCC TGGAAC AGCCCCAAC TGCTCG GATGCC CATCCTGGT GAC 689
Asn AsnSer TrpAsn SerProAsn CysSer AspAla HisProGly Asp
185 190 195
TCC AGTGGA GACAGC TCGGGCCTC AACGAC ACTTTT GGGACCACA CCT 737
Ser SerGly AspSer SerGlyLeu AsnAsp ThrPhe GlyThrThr Pro
200 205 210
GCT GCCGAG TACTTT GAACGTGGC GTGCTG CACCTC CACCAGAGC CAT 785
Ala AlaGlu TyrPhe GluArgGly ValLeu HisLeu HisGlnSer His
215 220 225
GGC ATCGAC GACCTG GGGCCTCCG CGG,TGGCAGCTC ACAGCCTGC CTG 833
Gly IleAsp AspLeu GlyProPro ArgTrp GlnLeu.ThrAlaCys Leu
230 235 240
GTG CTGGTC ATCGTG CTGCTCTAC TTCAGC CTCTGG AAGGGCGTG AAG 881
Val LeuVa1 IleVal LeuLeuTyr PheSer LeuTrp LysGlyVal 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 Ly's Val Val'Trp!Ile.~Thr Ala Thr Met Pro TyrVal Val
265 270 275
WO 93/2.628 PCT/US93/05179
,~ t,
52
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 Ile Asp
280 285 290
GGC ATC AGA GCA TAC CTG AGC GTT GAC TTC TAC CGG CTC TGC GAG GCG 1025
Gly Tle 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 Ile Asp Ala Ala Thr Gln 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 Ile 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 Ile Val Thr Thr Ser Ile.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 Va1 Val Phe Ser Phe Leu Gly Tyr Met Ala
360 365 370
CAG A~.G CAC AGT GTG CCC ATC GGG GAC GTG GCC AAG GAC GGG CCA GGG 1265
Gln Lys His Ser Val Pro Ile 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 Zle Phe Ile Ile Tyr Pro Glu Ala Ile 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 '~rp Ala Val Va1 ' Phe~ Phe 'Ile Nlet Leu Leu Thr Leu , Gly Ile
40S 410 415 420
WO 93/24628 PCT/US93/05179
v :A
. n ., ,.
53
GAC AGC GCC ATG GGT GGT,ATG GAG TCA GTG ATC ACC GGG CTC ATC GAT 1409
Asp Ser A1a Met Gly Gly Met Glu Ser Val Ile Thr Gly Leu Tle Asp
425 430 435
GAG TTC CAG CTG CTG CAC AGA CAC CGT GAG CTC TTC ACG CTC TTC ATC 1457
Glu Phe Gln Leu Leu His Arg His Arg Glu Leu Phe Thr Leu Phe Ile
440 445 450
GTC CTG GCG ACC TTC CTC CTG TCC CTG TTC TGC GTC ACC AAC GGT GGC 1505
Val Leu Ala Thr Pha 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
Ile Tyr Val Phe Thr Leu Leu Asp His Phe Ala Ala.Gly Thr Ser.Ile
470 475 480
CTC TTT GGAGTG CTCATC GAAGCC ATCGGAGTG GCCTGG TTCTAT GGT 1601
Leu Phe GlyVal LeuIle GluAla IleGlyVal AlaTrp PheTyr Gly
485 490 495 500
GTT GGG CAGTTC AGCGAC GACATC CAGCAGATG ACCGGG CAGCGG CCC 1649
Val Gly GlnPhe SerAsp AspIle GlnGlnMet ThrGly GlnArg Pro
5~5: 510 515
AGC CTG TACTGG CGGCTG TGCTGG AAGCTGGTC AGCCCC TGCTTT CTC 169?
Ser Leu TyrTrp ArgLeu CysTrp LysLeuVal SerPro CysPhe Leu
520 525 530
CTG: TTC GTGGTC GTGGTC AGCATT GTGACCTTC AGACCC CCCCAC TAC 1745
Leu Phe ValVa1 ValVal SerIle Va1ThrPhe ArgPro ProHis Tyr
535 540 545
GGA GCC TAGATC TTCCCC GACTGG GCCAACGCG CTGGGC TGGGTC ATC 1793
Gly Ala TyrI1'ePhePro 'Asp'Trp'Alal~snAla LeuGly TrpVal Ile
550 555 560
WO 93/24628 PCT/US93/OS179
. . -., n ~.. , t
~ I ~e ~~ .-
21308
54
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 Ile 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 G1u Lys Leu Ala Tyr Ala Ile Ala
585 590 595
CCC GAG AAG GAC CGT GAG CTG GTG GAC AGA GGG GAG GTG CGC CAG TTC 1937
Pro G1u Lys Asp Arg Glu Leu Val Asp Arg Gly G1u Va1 Arg Gln Phe
600 60S 610
ACG CTC CGC CAC TGG CTC AAG GTG TAGAGGGAGC AGAGACGAAG ACCCCAGGAA 1991
Thr Leu Arg His Trp Leu Lys Val
615 620
GTCATCCTGC AATGGGAGAGACACGAACAA TCTAAGTTTCGAGAGAAAGG 2051
ACCAAGGAAA
AGGGCAACTT CTACTCTTCAACCTCTACTGAAAACACAAACAACAAAGCAGAAGACTCCT 2111
CTCTTCTGAC TGTTTACACCTTTCCGTGCCGGGAGCGCACCTCGCCGTGTCTTGTGTTGC 2171
TGTAATARCG ACG.TAGATCTGTGCAGCGAGGTCCACCCCGTTGTTGTCCCTGCAGGGCAG 2231
AAAAACGTCT AACTTCATGCTGTCTGTGTGAGGCTCCCTCCCTCCCTGCTCCCTGCTCCC 2291
GGCTCTGAGG CTGCCCCAGGGGCACTGTGTTCTCAGGCGGGGATCACGATCCTTGTAGAC 2351
GCACCTGCTG AGAATCCCCGTGCTCACAGTAGCTTCCTAGACCATTTACTTTGCCCATAT 2411
TAAAAAGCCA AGTGTCCTGCTTGGTTTAGCTGTGCAGAAGGTGAAATGGAGGAAACCACA 2471
AATTCATGCA AAGTCCTTTC CCGATGCGTG-GCTCCCAGCA GAGGCCGTAA ATTGAGCGTT 2531
~ I ' j _ i'' . ,
CAGTTGACACATTGCACACACAGTCTGTTCAGAGGCATTG GAGGATGGGGGTCCTGGTAT 2591
GTCTCACCAGGAAATTCTGTTTATGTTCTTGCAGCAGAGA GAAATAAAACTCCTTGAAAC 2651
WO 93/24628 PCT/US93J05179
213608'
=,q~;.
CAGCTCAGGC TACTGCCACT CAGGCAGCCT GTGGGTCCTT GTGGTGTAGG GAACGGCCTG 2711
AGAGGAGCGT GTCCTATCCC CGGACGCATG CAGGGCCCCC ACAGGAGCGT GTCCTATCCCM 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 3077.
CCCCCACAGG AGCGTGTACTACCCCAGGACGCATGCAGGGCCCCCFsTGCAGGCAGCCTGC3131
AGACCAACAC TCTGCCTGGCCTTGAGCCGTGACCTCCAGGAAGGGACCCCACTGGAATTT3191
TATTTCTCTC AGGTGCGTGCCACATCAATAACAACAGTTTTTATGTTTGCGAATGGCTTT3251
TTAAAATCAT ATTTACCTGTGAATCAAAACAAATTCAAGAATGCAGTATCCGCGAGCCTG3311
CTTGCTGATA TTGCAGTTTTTGTTTACAAGAATAATTAGCAATACTGAGTGAAGGATGTT3371
GGCCAAAAGC TGCTTTCCATGGCACACTGCCCTCTGCCACTGACAGGAAAGTGGATGCCA3431
TAGTTTGAAT TCATGCCTCAAGTCGGTGGGCCTGCCTACGTGCTGCCCGAGGGCAGGGGC3491
CGTGCAGGGC CAGTCATGGC.TGTCCCCTGCAAGTGGACGTGGGCTCCAGGGACTGGAGTG3551
TAATGCTCGG TGGGAGCCGTCAGCCTGTGAACTGCCAGGCAGCTGCAGTTAGCACAGAGG3611
ATGGCTTCCC CATTGCCTTC TGGGGAGGGA CACAGAGGAC GGCTTCCCCA TCGCCTTCTG 3671
,. ; ~z ' ~ , ~ ,
GCCGCTGCAG TCAGCACAGA GAGCGGCTTC CCCATTGCCT TCTGGGGAGG GACACAGAGG 3731
ACAGTTTCCC CATCGCCTTC TGGTTGTTGA AGACAGCACA GAGAGCGGCT TCCCCATCGC 3791
WO 93/24628 P'C'1'/US93/OS179
.r
"~ , ~ y .a
56
CTTCTGGGGA GGGGCTCCGT GTAGCAACCC AGGTGTTGTC CGTGTCTGTT GACCAATCTC 3851
TATTCAGCAT CGTGTGGGTC CCTAAGCACA ATAAAAGACA TCCACAATGG AAA.AAAAAA.A 3911
AGGAATTC 3919
(2) INFORMATION FOR SEQ ID N0:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 620 amino acids
(B) TYPE: amino acid
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:2:
Met Ser Lys Ser Lys Cys Ser Val.Gly Leu Met Ser Ser Val Val Ala
1~ 15
Pro Ala Lys Glu Pro Asn Ala Va1 Gly Pro Lys Glu Val Glu Leu Ile
20 25 30
Leu Val Lys Glu Gln Asn Gly Va1 Gln Leu Thr Ser Ser Thr Leu Thr
35 40 45
Asn Pro Arg Gln Ser Pro Val Glu Ala Gln Asp Arg Glu Thr Trp Gly
50 55 60,
Lys Lys Ile Asp Phe Leu Leu Ser Val Ile Gly Phe Ala Val Asp Leu
65 70 75 80
Ala Asn Val Trp Arg Ph'e'Pro'Tyr~'Leu Cy$ Tyr'Lys Asn Gly,Gly Gly
85 90 95
Ala Phe Leu Val Pro Tyr Leu Leu Phe Met Val Ile Ala G1y Met Pro
100 105 110
WO 93/24628 ... PCI'/US93/OS179
'~ 'v
s7
Leu Phe Tyr Met Glu Leu Ala Leu Gly Gln Phe Asn Arg G1u Gly Ala
115 120 125 ",
Ala Gly Val Trp Lys Ile Cys Pro Ile Leu Lys Gly Val Gly Phe Thr
130 135 140
Val Ile Leu Ile Ser Leu Tyr Val Gly Phe Phe Tyr Asn Val Ile Ile
145 150 155 160
Ala Trp Ala Leu His Tyr Leu Phe Ser Ser Phe Thr Thr Glu Leu Pro
165 170 175
Trp Ile 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 G1y Va1 Leu His Leu
210 215 220
His Gln Ser His Gly Ile Asp Asp Leu Gly Pro Pro Arg Trp G1n Leu
225 230 235 240
Thr Ala Cys Leu Val Leu Val Ile Val Leu Leu Tyr Phe Ser Leu Trp
245 250 255
Lys Gly Val Lys Thr Ser Gly Lys Val Val Trp Ile 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 Ile Asp~Gly Ile Arg'Ala ~'yr Leu Ser Val Asp Phe Tyr Arg
290 295 300
Leu Cys Glu Ala Ser Val.Trp Ile Asp Ala Ala Thr Gln Val Cys Phe
305 310 315 320
,; , . ; , ... ,> .'.:; _.:: . . ;. ;''
WO 93/24628 . PC1"/US93/0~1'79
213 6 ~ 8'~ ~ P ~~:
58
Ser Leu Gly Val Gly Phe Gly Val Leu Ile Ala Phe Ser Ser Tyr Asn
325 330 335 ..
Lys Phe Thr Asn Asn Cys Tyr Arg Asp Ala Ile Va1 Thr Thr Ser Ile
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 Gln Lys His Ser Val Pro Ile Gly Asp Val Ala Lys
370 375 380
Asp Gly Pro Gly Leu Ile Phe Ile Ile Tyr Pro Glu Ala Ile Ala Thr
385 390 395 400
Leu Pro Leu Ser Ser Ala Trp Ala Val Val Phe Phe Ile Met ~Leu Leu
405 410 415
Thr Leu Gly Ile Asp Ser Ala filet Gly Gly Met G1u Ser Val Ile Thr
420 . 425 430
Gly Leu Ile Asp GIu Phe Gln Leu Leu His Arg His Arg Glu Leu Phe
435 440 445
Thr LeuPhe Ile Leu Ala Phe LeuSer LeuPhe Val
Val Thr Leu Cys
450 455 460
Thr AsnGly Gly Tyr Val Thr LeuAsp HisPhe Ala
Ile Phe Leu Ala
465 470 475 480
GIy ThrSer Ile Phe Gly Leu GluAla IleGly Ala
Leu Val Ile Val
485 490 495
Trp Phe Tyr Gly Val Gly'~Glri Phe''Ser Asp Asp ~~le Gln Gln,Met Thr
500 505 510
Gly Gln Arg Pro Ser Leu Tyr Trp Arg Leu Cys Trp Lys Leu Val Ser
515 520 525
WO 93/24628 PC.T/US93/U5179
~I~v': v: i: .. .:.
59
Pro Cys Phe Leu Leu Fhe Val Val Val Val Ser Ile Val Thr Phe Arg
530 535 540 ",
Pro Pro His Tyr G1y AIa Tyr Ile Phe Pro Asp Trp Ala Asn Ala Leu
545 550 555 560
Gly Trp Val Ile Ala Thr Ser Ser Met Ala Met Val Pro Tle 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 Ile Ala Pro Glu Lys Asp Arg Glu Leu Val Asp Arg Gly Glu
595 600 605
Val Arg Gln Phe Thr Leu Arg His Trp Leu Lys Val
610. 6~.5 620
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A)' LENGTH: 40 base pairs
(B)- TYPE: nucleic acid
(C) STRAN~RDNESS: double
(D) TOPOLOGY; linear
:(ii) MOLECULE TYPE: DNA
(iii) HYPOTHETICAL: XES
(iv) ANTI-SENSE: NO
(ix) FEATURE:
(~A) 'NAMEIKEY: ''- ' '1
(B) LOCATION: 1..40
(D) OTHER INFORMATION: /label= consensus
/note= "consensus sequence of VNTR element in 3'
untranslated region of HUDAT cDNA"
,. , .. .. .. : .. ' ~ ,.;; ;;
WO 93/24628 ' ' PCT/US93/U5179
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:3: M
AGGAGCGTGT ACTATCCCAG GACGCATGCA GGGCCCCCAC 40
(2) INFORMATION FOR SEQ ID N0:4:
(i) SEQTJENCE 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: x...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 N0:4:
TGTGGTGTAG GGAACGGCCT GAG
23
( 2 ) INFORMATION FOR SEA' ID NO : ~ : ' ~ ,
w ( i ) SEQUENCE CF~AR.ACTERISTICS
(A) LENGTH: 24 base pairs
(g) TYFE: nucleic acid
PCT/US93/05179
WO 93/24528
sz
(C) STRANDEDNESS: single
...
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (synthetic)
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(ix) FEATURE:
(A) NAMELY: -
(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: S:
CTTCCTGGAG GTCACGGCTC AAGG 24
