CA 02285690 1999-10-07
Gene Necessar~for Striatal Function and Uses Thereof
FIELD OF THE INVENTION
The present invention relate;; to polynucleotides expressed in the brain and
which are involved
in neurological disorders. Wore particularly, the present invention relates to
polynucleotides
expressed in the striatum, nucleus accumbens and olfactory tubercule, and
which are down-
regulated in the course of CAG repeat disorders, such as Huntington's disease.
The present
invention also describes variants and derivatives of these polynucleotides;
processes for
making these polynucleotides, and their agonists and antagonists, and uses of
these
polynucleotides, variants, derivatives, agonists and antagonists.
BACKGROUND OF THE INVENTION
Very few if any ei~ective treatments exist for neurological disorders
characterized by
progressive cell loss, known as neurodegenerative diseases, as well as those
involving acute
cell loss, such as stroke and trauma.
Huntington's disease (HD) is an inherited neurological disorder that is
transmitted in
autosomal dominant fashion. HD results from genetically programmed
degeneration of
neurons in certain areas of t',he brain. Huntington's disease is caused by a
mutation of the gene
IT IS that codes for the protein huntingtin. The huntingtin gene contains a
polymorphic
stretch of repeated CAG trinucleotides that encode a polyglutamine tract
within huntingtin. If
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this tract exceeds 35 in number, Huntington's disease results. Huntington's
disease is only
one of a number of neurological diseases which are characterised by these
polyglutamine
repeats (Ross, 1997). Schizophrenia, Alzheimer's disease, stroke, trauma, and
Parkinson's
disease also affect the basal ganglia.
Huntingtin has no sequence similarity to known proteins (Group THDCR, 1993;
Sisodia,
1998). The function of the normal or mutated HD form of huntingtin has not
been defined by
the prior art. It is evident, however, that the expression of the HD form of
huntingtin leads to
progressive and selective neuronal loss. It has been demonstrated that the
GABA- and
enkephalin-containing medium spiny projection neurons of the caudate-putamen
eventually die
as a result of HD (Richfield et al., 1994). Patients with minimal cell loss,
however, still
present with motor and cognitive symptoms suggesting that neuronal
dysfunction, and not
simply cell loss, contribute to the symptoms of HD. The motor symptoms of HD
include the
development of chorea, dysl:onia, bradykinesia and tremors (Young et al.,
1986). Voluntary
1 S movements may also be affected such that there may be disturbances in
speech (Ludlow et al.,
1987) and degradation of fine motor co-ordination (Young et al., 1986). In
addition to motor
decline, emotional disturbances and cognitive loss are also evident during the
progression of
HD (Came et al., 1978).
Despite the fact that huntin~;tin is ubiquitously expressed, ITD specifically
affects cells of the
basal ganglia, structures deep within the brain that have a number of
important functions,
including co-ordinating movement. The basal ganglia includes the caudate
nucleus, the
putamen, the nucleus accumbens and the olfactory tubercule. HD also affects
the brain's outer
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surface, or cortex, which controls thought, perception, and memory. The
mechanism by
which only a small group of neurons in the striatum and cortex are rendered
vulnerable to this
ubiquitously expressed mutant protein is not known. There are no effective
treatments for
Huntington's disease.
Huntington's disease is widf;ly believed to be a gain-of function disorder but
neither the
normal function nor the gained function of huntingtin is known. Because the
function for
huntingtin is not known, there is little insight into the disease process. It
was believed that
huntingtin was related to neuronal intranuclear inclusions (NII). However,
recent results have
cast doubt on our understanding of the role of the NII in Huntington's disease
(Saudou et al.,
1998) or in other CAG repeat disorders (Klement et al., 1998; see also
commentary by
Sisodia, 1998).
The development of a mouse carrying the S' end of the human Huntington's
disease gene (the
promoter and first exon; Mangiarini et al., 1996) was an important step in the
development of
the tools that will allow us to understand the function (and gain-of function)
associated with
huntingtin. R6/2 mice exhibit a rapidly progressing neurological phenotype
with onset at
about 8 weeks. This phenotype includes a movement disorder characterised by
shuddering,
resting tremor, epileptic sei:~ures and stereotyped behaviour. These symptoms
suggest that the
function of the basal ganglia is affected by the expression of the human exon
1 transgene prior
to neuronal cell death. By 12 weeks the affected mice have significantly
reduced brain weights
and they die by about 13 weeks of age. Neuronal intranuclear inclusions (NII)
develop at
about 4 weeks (Davies et al., 1997). As is observed in human Huntington's
disease patient,
the R6/2 mice show changes in neuronal receptors (Cha et al., 1998). The
present inventors
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have also demonstrated that changes in the expression of DARPP-32 and
cannabinoid
receptors change over time in HD mice; such changes have also been observed in
human
Huntington's disease patients (unpublished results). The loss of the
cannabinoid receptor is
one of the earliest documented changes that occur prior to neuronal
degeneration in human
HD patients. The R6/2 model, therefore, mimics the early phases of HD; a point
in disease
development where intervention would be most appropriate.
SUMMARY OF THE INVENTION
The present invention provides nucleotide sequences that identify and encode a
novel gene
(DHD) that is normally highly expressed in mammalian striatum, nucleus
accumbens and
olfactory tubercule. The invention teaches an isolated polynucleotide segment,
comprising a
polynucleotide sequence selected from the group consisting of (a) a sequence
comprising
SEQ ID NO:1; (b) a sequence comprising SEQ ID N0:2; (c) a sequence having
nucleotides
1140 to 3235 of SEQ ID NO:1; (d) a sequence which is at least 80% homologous
with a
sequence of (a), (b) or (c); ~(e) variants of (a), (b), (c) or (d), and; (~ a
sequence which
hybridizes to (a), (b), (c) or (d) under stringent conditions. In a preferred
embodiment, the
isolated polynucleotide segment is RNA. The invention also teaches an isolated
polynucleotide segment, which retains substantially the same biological
function or activity as
the polynucleotide encoded by the polynucleotide sequence.
Further preferred embodiments of the invention are polynucleotides that are at
least 70%
identical over their entire length to a polynucleotide encoding DHD
polypeptide or
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polynucleotide, and polynucleotides which are complementary to such
polynucleotides.
Alternatively, most highly preferred are polynucleotides that comprise a
region that is at least
80% identical over their entire length to a polynucleotide encoding DHD of SEQ
ID NO. and
polynucleotides complementary thereto. In this regard, polynucleotides at
least 90% identical
over their entire length to the same are particularly preferred, and among
these particularly
preferred polynucleotides, those with at least 95% are especially preferred.
Furthermore, those
with at least 97% are highly preferred among those with at least 95%, and
among these those
with at least 98% and at least 99% are particularly highly preferred, with at
least 99% being
the more preferred.
The invention also provides for a vector with the isolated polynucleotide
segment, and an
isolated host cell comprising; the vector. The invention also teaches a
process for producing a
polypeptide of a polynucleotide sequence comprising the step of culturing the
host cell under
conditions sufFlcient for the production of said polypeptide.
The invention further providles an isolated polynucleotide fragment of the
polynucleotide
sequence, wherein the polynucleotide sequence is selected from the group
consisting o~ (a) a
sequence having at least 1 S sequential bases of nucleotides 1140 to 323 5 of
SEQ ID NO: 1;
(b) a sequence having at least 30 sequential bases of nucleotides 1140 to 3235
of SEQ ID NO:
1;
(c) a sequence having at least 50 sequential bases of SEQ ID NO:1 or SEQ ID
NO: 2; (d) a
sequence which is at least 90% homologous with a sequence of (a), (b) or (c);
(e) variants of
(a), (b), (c) or (d), and; (fj a. sequence which hybridizes to (a), (b), (c)
or (d) under stringent
conditions.
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The invention also teaches a method for identifying a compound which inhibits
or promotes
the activity of a polynucleotide segment of claim 1, comprising the steps o~
(a) selecting a
control animal having said segment and a test animal having said segment; (b)
treating said test
animal using a compound; and (c) determining the relative quantity of RNA
corresponding to
said segment, as between said animals. In a preferred embodiment, the animal
is a mammal,
preferably a mouse, and preferably a transgenic mouse.
The invention also teaches a method for identifying a compound which inhibits
or promotes
the activity of a polynucleotide segment of claim 1, comprising the steps of
(a) selecting a
host cell of claim 4; (b) cloning said host cell and separating said clones
into a test group and
a control group; (c) treating said test group using a compound; and (c)
determining the
relative quantity of RNA corresponding to said segment, as between said test
group and said
control group.
The invention further teaches a method for diagnosing the presence of or the
predisposition
for a CAG repeat disorder, said method comprising determining the level of
expression of
RNA corresponding to the segments of claim 1 in an individual relative to a
predetermined
control level of expression, wherein a decreased expression of said RNA as
compared to said
control is indicative of a CAG repeat disorder. Preferably, the expression is
measured by in
situ hybridization, fluorescent in situ hybridization, polymerase chain
reaction, or DNA
fingerprinting technique. In a preferred embodiment, the CAG repeat disorder
is
Huntington's disease.
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BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 A is a portion of an autoradiogram of the differential display reaction
identifying DHD
in mouse brain mRNA.
FIB. 1B is a northern blot confirming that DHD has a lower steady-state level
of expression in
the striatum of transgenic HD mice.
FIG. 2 is a nucleotide sequence of the differential display cDNA fragment
pDHD.
FIG. 3A shows the in situ hybridization of probe 1 to coronal and saggital
brain sections of
10 week-old wild-type and HD mice.
FIG. 3B shows the in situ hybridization corresponding to spatial and temporal
expression of
DHD in brain sections of wild-type and HD mice over the period of time that
the HD mice
develop abnormal movements and postures.
FIG. 3C shows the in situ hybridization corresponding to expression of DHD in
brain sections
of one day old wild-type and HD mice.
FIG. 3D shows the in situ hybridization corresponding to distribution of the
mRNA of DHD
in mouse striatal neurons.
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FIG. 4 is the in situ hybridi~:ation corresponding to mRNA distribution of the
rat homologue
of DHD in rat brain tissue.
FIG. S shows a Southern blot analysis of DNA from wild-type and transgenic HD
mice
hybridized to the pDHD cDNA probe.
FIG. 6 is a nucleotide sequemce of cDHD-1, and corresponds to SEQ ID NO. 1.
FIG. 7 is a restriction map of cDHD-1.
FIG. 8 is a nucleotide segue-nce of cDHD-2, and corresponds to SEQ ID NO. 2.
FIG. 9 is a restriction map of cDHD-2.
1 S FIG. 10 is a schematic diagram showing the alignment of cDHD-1 and -2 and
the regions that
are identical and unique between the two clones.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS OF THE INVENTION
The following illustrative e~;planations are provided to facilitate
understanding of certain terms
used frequently herein. The. explanations are provided as a convenience and
are not limitative
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of the invention.
"Host cell" is a cell which h;as been transformed or transfected, or is
capable of transformation
or transfection by an exogenous polynucleotide sequence.
"Identity", "similarity" or "homologous", as used in the art, are
relationships between two or
more polynucleotide sequences, as determined by comparing the sequences. In
the art,
identity also means the degree of sequence relatedness between polynucleotide
sequences, as
the case may be, as determined by the match between strings of such sequences.
Both identity
and similarity can be readily calculated (Lesk, A. M., 1988; Smith, D. W.,
1993; Griffin, A.
M., and Grii~rn, H. G., 1994; von Heinje, G., 1987; and Gribskov, M. and
Devereux, J.,
1991). While there exist a number of methods to measure identity and
similarity between two
polynucleotide sequences, both terms are well known to skilled artisans (von
Heinje, G., 1987;
Gribskov, M. and Devereux, 1991; and Carillo, H., and Lipman, D., 1988).
Methods
commonly employed to detc;rmine identity or similarity between sequences
include, but are not
limited to those disclosed in Carillo, H., and Lipman, D. (1988). Methods to
determine
identity and similarity are codified in computer programs. Preferred computer
program
methods to determine identity and similarity between two sequences include,
but are not
limited to, GCG program package (Devereux, J., et al., 1984), BLASTP, BLASTN,
and
FASTA (Atschul, S. F. et al.., 1990).
"Isolated" means altered "by the hand of man" from its natural state; i.e.,
that, if it occurs in
nature, it has been changed or removed from its original environment, or both.
For example, a
naturally occurnng polynuc:leotide naturally present in a living organism in
its natural state is
CA 02285690 1999-10-07
not "isolated," but the same polynucleotide separated from coexisting
materials of its natural
state is "isolated", as the term is employed herein. As part of or following
isolation, such
polynucleotides can be joined to other polynucleotides, such as DNA, for
mutagenesis, to
form fusion proteins, and for propagation or expression in a host, for
instance. The isolated
polynucleotides, alone or joined to other polynucleotides such as vectors, can
be introduced
into host cells, in culture or iin whole organisms. Introduced into host cells
in culture or in
whole organisms, such DNA, still would be isolated, as the term is used
herein, because they
would not be in their naturally occurring form or environment. Similarly, the
polynucleotides
may occur in a composition, such as a media formulations, solutions for
introduction of
polynucleotides, for examplE;, into cells, compositions or solutions for
chemical or enzymatic
reactions, for instance, which are not naturally occurring compositions, and,
therein remain
isolated polynucleotides within the meaning of that term as it is employed
herein.
"Plasmids". Starting plasmids disclosed herein are either commercially
available, publicly
available, or can be constructed from available plasmids by routine
application of well known,
published procedures. Many plasmids and other cloning and expression vectors
that can be
used in accordance with the present invention are well known and readily
available to those of
skill in the art. Moreover, those of skill readily may construct any number of
other plasmids
suitable for use in the invention.
"Polynucleotides(s)" of the present invention may be in the form of RNA, such
as mRNA, or
in the form of DNA, including, for instance, cDNA and genomic DNA obtained by
cloning or
produced by chemical synthetic techniques or by a combination thereof. The DNA
may be
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double-stranded or single-stranded. Single-stranded polynucleotides may be the
coding strand,
also known as the sense strand, or it may be the non-coding strand, also
referred to as the anti-
sense strand. Polynucleotides generally refers to any polyribonucleotide or
polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or
DNA.
Thus, for instance, polynucleotides as used herein refers to, among others,
single-and double-
stranded DNA, DNA that is a mixture of single- and double-stranded regions or
single-,
double- and triple-stranded regions, single- and double-stranded RNA, and RNA
that is
mixture of single- and double-stranded regions, hybrid molecules comprising
DNA and RNA
that may be single-stranded or, more typically, double-stranded, or triple-
stranded, or a
mixture of single- and double-stranded regions. In addition, polynucleotide as
used herein
refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA.
The
strands in such regions may be from the same molecule or from different
molecules. The
regions may include all of one or more of the molecules, but more typically
involve only a
region of some of the molecules. One of the molecules of a triple-helical
region often is an
oligonucleotide. As used herein, the term polynucleotide also includes DNA or
DNA that
contain one or more modified bases. Thus, DNA or DNA with backbones modified
for
stability or for other reasons. are "polynucleotides" as that term is intended
herein. Moreover,
DNA or DNA comprising unusual bases, such as inosine, or modified bases, such
as tritylated
bases, to name just two examples, are polynucleotides as the term is used
herein. It will be
appreciated that a great variety of modifications have been made to DNA and
RNA that serve
many useful purposes known to those of skill in the art. The term
polynucleotide as it is
employed herein embraces such chemically, enzymatically or metabolically
modified forms of
polynucleotides, as well as the chemical forms of DNA and RNA characteristic
of viruses and
cells, including simple and complex cells, inter alia. Polynucleotides
embraces short
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polynucleotides often referred to as oligonucleotide(s). It will also be
appreciated that RNA
made by transcription of this. doubled stranded nucleotide sequence, and an
antisense strand of
a nucleic acid molecule of the invention or an oligonucleotide fragment of the
nucleic acid
molecule, are contemplated within the scope of the invention. An antisense
sequence is
constructed by inverting the sequence of a nucleic acid molecule of the
invention, relative to
its normal presentation for transcription. Preferably, an antisense sequence
is constructed by
inverting a region preceding the initiation codon or an unconserved region.
The antisense
sequences may be constructed using chemical synthesis and enzymatic ligation
reactions using
procedures known in the art.
"Stringent hybridization conditions" are those which are stringent enough to
provide
specificity, reduce the number of mismatches and yet are sufficiently flexible
to allow
formation of stable hybrids at an acceptable rate. Such conditions are known
to those skilled in
the art and are described, fo:r example, in Sambrook, et al, ( 1989). By way
of example only,
1 S stringent hybridization with short nucleotides may be earned out at 5-
10° below the TM using
high concentrations of probe; such as 0.01-1.0 pmole/ml. Preferably, the term
"stringent
conditions" means hybridization will occur only if there is at least 95% and
preferably at least
97% identity between the sequences.
"Variant(s)" of polynucleotides are polynucleotides that differ in nucleotide
sequence from
another, reference polynucle;otide. Generally, differences are limited so that
the nucleotide
sequences of the reference a.nd the variant are closely similar overall and,
in many regions,
identical. Changes in the nucleotide sequence of the variant may be silent.
That is, they may
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not alter the amino acids encoded by the polynucleotide. Where alterations are
limited to
silent changes of this type a variant will encode a polypeptide or
polynucleotide with the same
amino acid sequence as the reference. Changes in the nucleotide sequence of
the variant may
alter the amino acid sequence of a polypeptide encoded by the reference
polynucleotide. Such
nucleotide changes may result in amino acid substitutions, additions,
deletions, fusions and
truncations in the polypeptid.e or polynucleotide encoded by the reference
sequence.
As hereinbefore mentioned, the present inventors have identified and sequenced
a DNA
sequence encoding DHD. The DNA sequence is shown in the Sequence Listing as
SEQ ID
NO:1 and N0:2.
It will be appreciated that the invention includes nucleotide or amino acid
sequences which
have substantial sequence homology with the nucleotide sequences shown in the
Sequence
Listing as SEQ ID NO:1 and N0:2. The term "sequences having substantial
sequence
homology" means those nucleotide and amino acid sequences which have slight or
inconsequential sequence variations from the sequences disclosed in the
Sequence Listing as
SEQ ID NO:1 and N0:2 i.e. the homologous sequences function in substantially
the same
manner to produce substantially the same polypeptides as the actual sequences.
The variations
may be attributable to local mutations or structural modifications. It is
expected that a
sequence having 85-90% sequence homology with the DNA sequence of the
invention will
provide a functional DHD polypeptide. Nucleic acid sequences having
substantial sequence
homology also include nucleic acid sequences having at least 70%, preferably
at least 80%
homology with the nucleic acid sequence as shown in SEQ. ID. NO:1 and N0:2;
and
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fragments thereof having at least 15 to 50, preferably at least 15 bases, and
preferably 20 to
30, which will hybridize to these sequences under stringent hybridization
conditions.
The polynucleotides of the present invention may be employed as research
reagents and
materials for discovery of treatments of and diagnostics for disease,
particularly human
disease, as further discussed herein.
Analysis of the complete nucleotide and amino acid sequences of the protein of
the invention
using the procedures of Sambrook et al., supra, will be used to determine the
expressed
region, initiation codon and untranslated sequences of the DI-ID gene. The
transcription
regulatory sequences of the gene may be determined by analyzing fragments of
the DNA for
their ability to express a reporter gene such as the bacterial gene lacZ.
Primer extension using
reverse transcriptase may also be used to determine the initiation site.
The nucleic acid molecules of the invention allow those skilled in the art to
construct
nucleotide probes for use in the detection of nucleotide sequences in
biological materials. As
shown in Figures 7 and 9 , a number of unique restriction sequences for
restriction enzymes
are incorporated in the nucleic acid molecule identified in the Sequence
Listing as SEQ ID
NO:1 and NO: 2, and these provide access to nucleotide sequences which code
for
polypeptides unique to the DHI~ polypeptide of the invention. Nucleotide
sequences unique
to DHD or isoforms thereof=, can also be constructed by chemical synthesis and
enzymatic
ligation reactions earned out: by procedures known in the art.
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A nucleotide probe may be Labeled with a detectable marker such as a
radioactive label which
provides for an adequate signal and has sufficient half life such as 32p, 3H,
14C or the like.
Other detectable markers which may be used include antigens that are
recognized by a specific
labeled antibody, fluorescent: compounds, enzymes, antibodies specific for a
labeled antigen,
and chemiluminescent compounds. An appropriate label may be selected having
regard to the
rate of hybridization and binding of the probe to the nucleotide to be
detected and the amount
of nucleotide available for hybridization. The nucleotide probes may be used
to detect genes
related to or analogous to DHD of the invention.
Accordingly, the present invention also provides a method of detecting the
presence of nucleic
acid molecules encoding a p~olypeptide related to or analogous to DHD in a
sample comprising
contacting the sample under hybridization conditions with one or more of the
nucleotide
probes of the invention labeled with a detectable marker, and determining the
degree of
hybridization between the nucleic acid molecule in the sample and the
nucleotide probes.
Hybridization conditions which may be used in the method of the invention are
known in the
art and are described for example in Sambrook J, et al., supra. The
hybridization product may
be assayed using techniques known in the art. The nucleotide probe may be
labeled with a
detectable marker as described herein and the hybridization product may be
assayed by
detecting the detectable marker or the detectable change produced by the
detectable marker.
The nucleic acid molecule of the invention also permits the identification and
isolation, or
synthesis of nucleotide sequences which may be used as primers to amplify a
polynucleotide
molecule of the invention, for example in polymerase chain reaction (PCR). The
length and
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bases of the primers for use in the PCR are selected so that they will
hybridize to different
strands of the desired sequence and at relative positions along the sequence
such that an
extension product synthesized from one primer when it is separated from its
template can
serve as a template for extension of the other primer into a nucleic acid of
defined length.
S
Primers which may be used in the invention are oligonucleotides i.e. molecules
containing two
or more deoxyribonucleotides of the nucleic acid molecule of the invention
which occur
naturally as in a purified restriction endonuclease digest or are produced
synthetically using
techniques known in the art such as, for example, phosphotriester and
phosphodiester methods
(See Good et al, 1977) or automated techniques (see, for example, Conolly, B.
A., 1987).
The primers are capable of acting as a point of initiation of synthesis when
placed under
conditions which permit the synthesis of a primer extension product which is
complementary
to the DNA sequence of the invention e.g. in the presence of nucleotide
substrates, an agent
for polymerization such as DNA polymerase and at suitable temperature and pH.
Preferably,
the primers are sequences that do not form secondary structures by base
pairing with other
copies of the primer or sequences that form a hair pin configuration. The
primer may be single
or double-stranded. When the primer is double-stranded it may be treated to
separate its
strands before using it to prepare amplification products. The primer
preferably contains
between about 7 and 25 nucleotides.
The primers may be labeled with detectable markers which allow for detection
of the amplified
products. Suitable detectable markers are radioactive markers such as P-32, S-
35, I-125, and
H-3, luminescent markers such as chemiluminescent markers, preferably luminol,
and
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Iluorescent markers, preferably dansyl chloride, fluorcein-5-isothiocyanate,
and 4-fluor-7-
nitrobenz-2-axa-1,3 diazole, enzyme markers such as horseradish peroxidase,
alkaline
phosphatase, .beta.-galactosidase, acetylcholinesterase, or biotin.
It will be appreciated that the primers may contain non-complementary
sequences provided
that a sufficient amount of the primer contains a sequence which is
complementary to a nucleic
acid molecule of the invention or oligonucleotide sequence thereof, which is
to be amplified.
Restriction site linkers may also be incorporated into the primers allowing
for digestion of the
amplified products with the appropriate restriction enzymes facilitating
cloning and sequencing
of the amplified product.
Thus, a method of determining the presence of a nucleic acid molecule having a
sequence
encoding DHD or a predetermined oligonucleotide fragment thereof in a sample,
is provided
comprising treating the sample with primers which are capable of amplifying
the nucleic acid
molecule or the predetermined oligonucleotide fragment thereof in a polymerase
chain reaction
to form amplified sequences., under conditions which permit the formation of
amplified
sequences and, assaying for amplified sequences.
The polymerase chain reaction refers to a process for amplifying a target
nucleic acid sequence
as generally described in Innis et al, Academic Press, 1989, in Mullis el al.,
U.S. Pat. No.
4,863,195 and Mullis, U.S. Pat. No. 4,683,202 which are incorporated herein by
reference.
Conditions for amplifying a nucleic acid template are described in M. A. Innis
and D. H.
Gelfand, 1989, which is also incorporated herein by reference.
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The amplified products can be isolated and distinguished based on their
respective sizes using
techniques known in the art. For example, after amplification, the DNA sample
can be
separated on an agarose gel and visualized, after staining with ethidium
bromide, under ultra
violet (UV) light. DNA may be amplified to a desired level and a further
extension reaction
may be performed to incorporate nucleotide derivatives having detectable
markers such as
radioactive labeled or biotin labeled nucleoside triphosphates. The primers
may also be labeled
with detectable markers. The detectable markers may be analyzed by restriction
and
electrophoretic separation or other techniques known in the art.
The conditions which may bE; employed in the methods of the invention using
PCR are those
which permit hybridization and amplification reactions to proceed in the
presence of DNA in a
sample and appropriate complementary hybridization primers. Conditions
suitable for the
polymerise chain reaction are generally known in the art. For example, see M.
A. Innis and D.
H. Gelfand, 1989, which is incorporated herein by reference. Preferably, the
PCR utilizes
polymerise obtained from the thermophilic bacterium Thermus aquatics (Taq
polymerise,
GeneAmp Kit, Perkin Elmer Cetus) or other thermostable polymerise may be used
to amplify
DNA template strands.
It will be appreciated that other techniques such as the Ligase Chain Reaction
(LCR) and
Nucleic-Acid Sequence Based Amplification (NASBA) may be used to amplify a
nucleic acid
molecule of the invention. In LCR, two primers which hybridize adjacent to
each other on the
target strand are ligated in the presence of the target strand to produce a
complementary
strand (Barney, 1991 and European Published Application No. 0320308, published
Jun. 14,
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1989). NASBA is a continuous amplification method using two primers, one
incorporating a
promoter sequence recognized by an RNA polymerase and the second derived from
the
complementary sequence of the target sequence to the first primer (U.S. Ser.
No. 5,130,238 to
Malek).
The present invention also teaches vectors which comprise a polynucleotide or
polynucleotides
of the present invention, host cells which are genetically engineered with
vectors of the
invention and the production of polynucleotides of the invention by
recombinant techniques.
In accordance with this aspect of the invention the vector may be, for
example, a plasmid
vector, a single or double-stranded phage vector, a single or double-stranded
RNA or DNA
viral vector. In certain preferred embodiments in this regard, the vectors
provide for specific
expression. Such specific expression may be inducible expression or expression
only in certain
types of cells or both inducible and cell-specific. Particularly preferred
among inducible
vectors are vectors that can be induced for expression by environmental
factors that are easy
to manipulate, such as temperature and nutrient additives. A variety of
vectors suitable to this
aspect of the invention, including constitutive and inducible expression
vectors for use in
prokaryotic and eukaryotic hosts, are well known and employed routinely by
those of skill in
the art. Such vectors includE:, among others, chromosomal, episomal and virus-
derived
vectors, e.g., vectors derived from bacterial plasmids, from bacteriophage,
from transposons,
from yeast episomes, from insertion elements, from yeast chromosomal elements,
from viruses
such as baculoviruses, papova viruses, such as S V40, vaccinia viruses,
adenoviruses, fowl pox
viruses, pseudorabies viruses and retroviruses, and vectors derived from
combinations thereof,
such as those derived from plasmid and bacteriophage genetic elements, such as
cosmids and
CA 02285690 1999-10-07
phagemids, all may be used for expression in accordance with this aspect of
the present
invention.
The following vectors, which are commercially available, are provided by way
of example.
Among vectors preferred for use in bacteria are pQE70, pQE60 and pQE-9,
available from
Qiagen; pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNHl6a,
pNHl8A,
pNH46A, available from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540,
pRITS
available from Pharmacia, and pBR322 (ATCC 37017). Among preferred eukaryotic
vectors
are pWLNEO, pSV2CAT, pOG44, pXTI and pSG available from Stratagene; and pSVK3,
pBPV, pMSG and pSVL available from Pharmacia. These vectors are listed solely
by way of
illustration of the many commercially available and well known vectors that
are available to
those of skill in the art for us,e in accordance with this aspect of the
present invention. It will
be appreciated that any other plasmid or vector suitable for, for example,
introduction,
maintenance, propagation or expression of a polynucleotide or polypeptide of
the invention in
a host may be used in this aspect of the invention. Generally, any vector
suitable to maintain,
propagate or express polynucleotides to express a polypeptide or
polynucleotide in a host
may be used for expression in this regard.
The appropriate DNA sequence may be inserted into the vector by any of a
variety of well-
known and routine techniques. In general, expression constructs will contain
sites for
transcription initiation and termination, and, in the transcribed region, a
ribosome binding site
for translation. The coding portion of the mature transcripts expressed by the
constructs will
include a translation initiating AUG at the beginning and a termination codon
appropriately
21
CA 02285690 1999-10-07
positioned at the end of the polynucleotide to be translated.
The DNA sequence in the e~;pression vector is operatively linked to
appropriate expression
control sequence(s), including, for instance, a promoter to direct mRNA
transcription.
Promoter regions can be selected from any desired gene using vectors that
contain a reporter
transcription unit lacking a promoter region, such as a chloramphenicol acetyl
transferase
("CAT") transcription unit, downstream of restriction site or sites for
introducing a candidate
promoter fragment; i.e., a fragment that may contain a promoter. As is well
known,
introduction into the vector of a promoter-containing fragment at the
restriction site upstream
of the cat gene engenders production of CAT activity, which can be detected by
standard CAT
assays. Vectors suitable to this end are well known and readily available,
such as pKK232-8
and pCM7. Promoters for expression of polynucleotides of the present invention
include not
only well known and readily available promoters, but also promoters that
readily may be
obtained by the foregoing technique, using a reporter gene. Amang known
prokaryotic
promoters suitable for expression of polynucleotides and polypeptides in
accordance with the
present invention are the E. coli lacI and lacZ and promoters, the T3 and T7
promoters, the
gpt promoter, the lambda Ply, PL promoters and the trp promoter. Among known
eukaryotic
promoters suitable in this regard are the CMV immediate early promoter, the
HSV thymidine
kinase promoter, the early and late SV40 promoters, the promoters of
retroviral LTRs, such as
those of the Rous sarcoma virus ("RSV"), and metallothionein promoters, such
as the mouse
metallothionein-I promoter.
Vectors for propagation and expression generally will include selectable
markers and
amplification regions, such as, for example, those set forth in Sambrook et
al., supra.
22
CA 02285690 1999-10-07
As hereinbefore mentioned, 'the present invention also teaches host cells
which are genetically
engineered with vectors of the invention.
Polynucleotide constmcts in host cells can be used in a conventional manner to
produce the
gene product encoder by the recombinant sequence. The DHD polynucleotide or
polypeptide
products or isoforms or parts thereof, may be obtained by expression in a
suitable host cell
using techniques known in the art. Suitable host cells include prokaryotic or
eukaryotic
organisms or cell lines, for example bacterial, mammalian, yeast, or other
fungi, viral, plant or
insect cells. Method;. for transforming or transfecting cells to express
foreign DNA are well
known in the art (See: for example, Itakura et al., U.S. Pat. No. 4,704,362;
Hinnen et al., 1978;
Murray et al., U.S. Pat. No. 4,801,542; Upshall et al., U.S. Pat. No.
4,935,349; Hagen et al.,
U.S. Pat. No. 4,784,~~50; A~;el et al., U.S. Pat. No. 4,399,216; Goeddal et
al., U.S. Pat. No.
4,766,075; and Sambrook et al, 1989, all of which are incorporated herein by
reference).
1 S Representative examples of appropriate hosts include bacterial cells, such
as streptococci,
staphylococci, E. coli, streptomyces and Bacillus subtilis cells; fungal
cells, such as yeast cells
and Aspergillus cells: insect cells such as Drosophila S2 and Spodoptera S~
cells; animal cells
such as CHO, COS, HeLa, C127, 3T3, BHK, 293 and Bowes melanoma cells; and
plant cells.
Host cells can be genetically engineered to incorporate polynucleotides and
express
polynucleotides of the present invention. Introduction of a polynucleotides
into the host cell
can be affected by calcium phosphate transfection, DEAF-dextran mediated
transfection,
transvection, microinjection, cationic lipid-mediated transfection,
electroporation,
23
CA 02285690 1999-10-07
transduction, scrape loading, ballistic introduction, infection or other
methods. Such methods
are described in many standard laboratory manuals, such as Davis et al. (1986)
and Sambrook
et al. ( 1989).
As hereinbefore mentioned, the present invention also teaches the production
of
polynucleotides of the invention by recombinant techniques.
The DHD polynucleotides may encode a polypeptide which is the mature protein
plus
additional amino or carboxyl-terminal amino acids, or amino acids interior to
the mature
a
polypeptide (when the mature form has more than one polypeptide chain, for
instance). Such
sequences may play a role in processing of a protein from precursor to a
mature form, may
allow protein transport, may lengthen or shorten protein half life or may
facilitate manipulation
of a protein for assay or production, among other things. As generally is the
case in vivo, the
additional amino acids may be processed away from the mature protein by
cellular enzymes.
A precursor protein, having 'the mature form of the polypeptide fused to one
or more
prosequences may be an inactive form of the polypeptide. When prosequences are
removed
such inactive precursors generally are activated. Some or all of the
prosequences may be
removed before activation. Generally, such precursors are called proproteins.
In sum, a polynucleotide of the present invention may encode a mature protein,
a mature
protein plus a leader sequence (which may be referred to as a preprotein), a
precursor of a
mature protein having one or more prosequences which are not the leader
sequences of a
preprotein, or a preproprotein, which is a precursor to a proprotein, having a
leader sequence
24
CA 02285690 1999-10-07
and one or more prosequences, which generally are removed during processing
steps that
produce active and mature forms of the polypeptide.
The polypeptides of the invention may be prepared by culturing the host/vector
systems
described above, in order to express the recombinant polypeptides.
Recombinantly produced
DHD based protein or parts thereof, may be further purified using techniques
known in the art
such as commercially available protein concentration systems, by salting out
the protein
followed by dialysis, by ai~mity chromatography, or using anion or cation
exchange resins.
Mature proteins can be expressed in mammalian cells, yeast, bacteria, or other
cells under the
control of appropriate promoters. Cell-free translation systems can also be
employed to
produce such proteins using DNA derived from the DNA constructs of the present
invention.
Appropriate cloning and expression vectors for use with prokaryotic and
eukaryotic hosts are
described by Sambrook et al., supra.
Polynucleotides of the invention, encoding the heterologous structural
sequence of a
polynucleotide or polypeptide of the invention generally will be inserted into
a vector using
standard techniques so that it is operably linked to the promoter for
expression. The
polynucleotide will be positioned so that the transcription start site is
located appropriately 5'
to a ribosome binding site. 7.'he ribosome binding site will be 5' to the AUG
that initiates
translation of the polynucleotide or polypeptide to be expressed. Generally,
there will be no
other open reading frames that begin with an initiation codon, usually AUG,
and lie between
the ribosome binding site and the initiation codon. Also, generally, there
will be a translation
CA 02285690 1999-10-07
stop codon at the end of the expressed polynucleotide and there will be a
polyadenylation
signal in constructs for use in eukaryotic hosts. Transcription termination
signal appropriately
disposed at the 3' end of the transcribed region may also be included in the
polynucleotide
construct.
S
For secretion of the translated protein into the lumen of the endoplasmic
reticulum, into the
pen-iplasmic space or into the extracellular environment, appropriate
secretion signals may be
incorporated into the expressed polynucleotide or polypeptide. These signals
may be
endogenous to the polynucleotide or they may be heterologous signals.
Microbial cells
employed in expression of proteins can be disrupted by any convenient method,
including
freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing
agents, such
methods are well know to those skilled in the art. DHD polynucleotide or
polypeptide can be
recovered and purified from recombinant cell cultures by well-known methods
including
ammonium sulfate or ethanol precipitation, acid extraction, anion or cation
exchange
chromatography, phosphocellulose chromatography, hydrophobic interaction
chromatography,
affinity chromatography, hydroxylapatite chromatography and lectin
chromatography. Most
preferably, high performance liquid chromatography is employed for
purification. Well known
techniques for refolding protein may be employed to regenerate active
conformation when the
polynucleotide is denatured during isolation and or purification.
In a preferred embodiment, a nucleic acid molecule of the invention may be
cloned into a
glutathione S-transferase (GST) gene fusion system for example the pGEX-1 T,
pGEX-2T
and pGEX-3X of Pharmacia. The fizsed gene may contain a strong lac promoter,
inducible to a
high level of expression by IPTG, as a regulatory element. Thrombin or factor
Xa cleavage
26
CA 02285690 1999-10-07
sites may be present which allow proteolytic cleavage of the desired
polypeptide from the
fusion product. The glutathiane S-transferase-DHD fusion protein may be easily
purified using
a glutathione sepharose 4B column, for example from Pharmacia. The 26 kd
glutathione S-
transferase polypeptide can be cleaved by thrombin (pGEX-1 or pGEX-2T) or
factor Xa
(pGEX-3X) and resolved from the using the polypeptide using the same affinity
column.
Additional chromatographic steps can be included if necessary, for example
Sephadex or
DEAE cellulose. The two enzymes may be monitored by protein and enzymatic
assays and
purity may be confirmed using SDS-PAGE.
The DHD protein or parts thereof may also be prepared by chemical synthesis
using
techniques well known in the: chemistry of proteins such as solid phase
synthesis (Mernfield,
1964) or synthesis in homogenous solution (Houbenweyl, 1987).
Within the context of the present invention, DHD polypeptide includes various
structural
forms of the primary protein which retain biological activity. For example,
DHD polypeptide
may be in the form of acidic or basic salts or in neutral form. In addition,
individual amino acid
residues may be modified by oxidation or reduction. Furthermore, various
substitutions,
deletions or additions may bf; made to the amino acid or nucleic acid
sequences, the net effect
being that biological activity of DHD is retained. Due to code degeneracy, for
example, there
may be considerable variation in nucleotide sequences encoding the same amino
acid.
The polypeptide may be expressed in a modified form, such as a fusion protein,
and may
include not only secretion signals but also additional heterologous functional
regions. Thus,
27
CA 02285690 1999-10-07
for instance, a region of additional amino acids, particularly charged amino
acids, may be
added to the C- or N-terminus of the polypeptide to improve stability and
persistence in the
host cell, during purification or during subsequent handling and storage.
Also, fusion proteins
may be added to the polynucleotide or polypeptide to facilitate purification.
Such regions may
be removed prior to final preparation of the polynucleotide or polypeptide.
The addition of
peptide moieties to polynucleotide or polypeptides to engender secretion or
excretion, to
improve stability or to facilitate purification, among others, are familiar
and routine techniques
in the art. In drug discovery, for example, proteins have been fused with
antibody Fc portions
for the purpose of high-throughput screening assays to identify antagonists
(see Bennett et al.,
1995, and Johanson et al.,1995).
This invention is also related to the use of the DHD polynucleotides to detect
complementary
polynucleotides as a diagnostic reagent.
1 S Detection of the level of expression of DHD in a eukaryote, particularly a
mammal, and
especially a human, will provide a diagnostic method for diagnosis of a
disease. Eukaryotes
(herein also "individual(s)"), particularly mammals, and especially humans,
exhibiting
decreased levels of DHD may be detected by a variety of techniques. Nucleic
acids for
diagnosis may be obtained from an infected individual's cells and tissues,
such as the striatum,
nucleus accumbens and olfactory tubercule. RNA may be used directly for
detection or may
be amplified enzymatically by using PCR (Saiki et al., 1986) prior to
analysis. As an example,
PCR primers complementan~ to the nucleic acid encoding DHD can be used to
identify and
analyze DHD presence and/or expression. Using PCR, characterization of the
level of DHD
present in the individual may be made by comparative analysis.
28
CA 02285690 1999-10-07
The invention thus provides a process for diagnosing disease by using methods
known in the
art and methods described herein to detect decreased expression of DHD
polynucleotide. For
example, decreased expression of DHD polynucleotide can be measured using any
on of the
methods well known in the art for the quantification of polynucleotides, such
as, for example,
PCR, RT-PCR, DNAse protection, northern blotting and other hybridization
methods. Thus,
the present invention provides a method for diagnosing triplet-repeat
disorders, and a method
for diagnosing a genetic pre--disposition for triplet-repeat disorders and
other disorders of the
basal ganglia including schizophrenia, stroke, trauma, Parkinson's disease and
Alzheimer's
disease (AD). More generally, the present invention provides a method for
diagnosing a
genetic pre-disposition for neurological disorders characterized by
progressive cell loss.
The invention also provides a method of screening compounds to identify those
which enhance
(agonist) or block (antagonist) the action of DHD polypeptides or
polynucleotides, such as its
interaction with DHD-binding molecules. The identification of mutations in
specific genes in
inherited neurodegenerative disorders, combined with advances in the field of
transgenic
methods, provides those of skill in the art with the information necessary to
further study
human diseases. This is extraordinarily usefizl in modeling familial forms of
triplet-repeat
disorders and other disorders of the basal ganglia including schizophrenia,
stroke, trauma,
Parkinson's disease and Alzheimer's disease (AD). More generally, the present
invention is
usefirl for modeling neurological disorders characterized by progressive cell
loss, as well as
those involving acute cell loss, such as stroke and trauma.
29
CA 02285690 1999-10-07
For example, to screen for agonists or antagonists, a synthetic reaction mix,
a cellular
compartment, such as a membrane, cell envelope or cell wall, or a preparation
of any thereof,
may be prepared from a cell that expresses a molecule that binds DHI~. The
preparation is
incubated with labeled DHD in the absence or the presence of a candidate
molecule which may
be a DHI~ agonist or antagonist. The ability of the candidate molecule to bind
the binding
molecule is reflected in decreased binding of the labeled ligand.
DHD-like erects of potential agonists and antagonists may by measured, for
instance, by
determining activity of a reporter system following interaction of the
candidate molecule with
a cell or appropriate cell preparation, and comparing the effect with that of
DHD or molecules
that elicit the same effects as DHD. Reporter systems that may be useful in
this regard include,
but are not limited to, colorimetric labeled substrate converted into product,
a reporter gene
that is responsive to changes in DHD activity, and binding assays known in the
art.
Another example of an assay for DHD antagonists is a competitive assay that
combines DHD
and a potential antagonist with membrane-bound DID-binding molecules,
recombinant DHI~
binding molecules, natural substrates or ligands, or substrate or ligand
mimetics, under
appropriate conditions for a competitive inhibition assay. DI-ID can be
labeled, such as by
radioactivity or a colorimetri.c compound, such that the number of DHD
molecules bound to a
binding molecule or converted to product can be determined accurately to
assess the
effectiveness of the potential antagonist.
Potential antagonists include small organic molecules, peptides, polypeptides
and antibodies
CA 02285690 1999-10-07
that bind to a polynucleotide or polypeptide of the invention and thereby
inhibit or extinguish
its activity. Potential antagonists also may be small organic molecules, a
peptide, a polypeptide
such as a closely related protein or antibody that binds the same sites on a
binding molecule,
such as a binding molecule, 'Nithout inducing DHD-induced activities, thereby
preventing the
action of DI-~ by excluding DID from binding.
Potential antagonists include a small molecule which binds to and occupies the
binding site of
the polypeptide thereby preventing binding to cellular binding molecules, such
that normal
biological activity is prevented. Examples of small molecules include but are
not limited to
small organic molecules, peptides or peptide-like molecules. Other potential
antagonists
include antisense molecules see Okano, 1988, for a description of these
molecules). Preferred
potential antagonists include compounds related to and derivatives of DHD.
Thus, the present invention provides a method for screening and selecting
compounds which
promote triplet-repeat disorders, and a method for screening and selecting
compounds which
treat or inhibit triplet-repeat disorders, as well as schizophrenia, stroke,
trauma, Parkinson's
disease and Alzheimer's disease. More generally, the present invention
provides a method for
screening and selecting compounds which promote or inhibit neurological
disorders
characterized by progressive; cell loss, as well as those involving acute cell
loss, such as stroke
and trauma.
The selected antagonists and agonists may be administered, for instance, to
inhibit progressive
31
CA 02285690 1999-10-07
and acute neurological disorders, such as Huntington's disease, schizophrenia,
Alzheimer's
disease (AD), stroke or trauma.
Antagonists and agonists and other compounds of the present invention may be
employed
alone or in conjunction with other compounds, such as therapeutic compounds.
The
pharmaceutical compositions. may be administered in any effective, convenient
manner
including, for instance, administration by direct microinjection into the
affected area, or by
intravenous or other routes. These compositions of the present invention may
be employed in
combination with a non-sterile or sterile carrier or carriers for use with
cells, tissues or
organisms, such as a pharmaceutical carrier suitable for administration to a
subject. Such
compositions comprise, for instance, a media additive or a therapeutically
effective amount of
antagonists or agonists of the invention and a pharmaceutically acceptable
carrier or excipient.
Such carriers may include, but are not limited to, saline, buffered saline,
dextrose, water,
glycerol, ethanol and combinations thereof. The formulation is prepared to
suit the mode of
administration.
The invention further provides diagnostic and pharmaceutical packs and kits
comprising one
or more containers filled with one or more of the ingredients of the
aforementioned
compositions of the invention. Associated with such containers) can be a
notice in the form
prescribed by a governmental agency regulating the manufacture, use or sale of
pharmaceuticals or biological products, reflecting approval by the agency of
the manufacture,
use or sale of the product for human administration.
The pharmaceutical compositions generally are administered in an amount
effective for
32
CA 02285690 1999-10-07
treatment or prophylaxis of a. specific indication or indications. It is
appreciated that optimum
dosage will be determined by standard methods for each treatment modality and
indication,
taking into account the indication, its severity, route of administration,
complicating conditions
and the like. In therapy or a;; a prophylactic, the active agent may be
administered to an
individual as an injectable composition, for example as a sterile aqueous
dispersion, preferably
isotonic. For administration to mammals, and particularly humans, it is
expected that the daily
dosage level of the active agent will be from 0.001 mg/kg to 10 mg/kg,
typically around 0.01
mg/kg. The physician in any event will determine the actual dosage which will
be most suitable
for an individual and will vary with the age, weight and response of the
particular individual.
The above dosages are exemplary of the average case. There can, of course, be
individual
instances where higher or lower dosage ranges are merited, and such are within
the scope of
this invention.
EXAMPLES
The present invention is further described by the following examples. These
examples, while
illustrating certain specific aspects of the invention, do not portray the
limitations or
circumscribe the scope of the disclosed invention.
EXAMPLE 1 - Isolation of DHD
Wild-type (B6CBAF1) and l-ID transgenic [B6CBA-TgN(Hdexonl)62Gpb] mice
(Jackson
Laboratories) and adult Sprague-Dawley rats (250-300 g; Charles River
Laboratories) and
33
CA 02285690 1999-10-07
were used in this study. The genotype of the mice was determined by PCR
amplification of a
100 by region of the integrated human HD exon 1 transgene using primers
corresponding to
nts 3340-3459 (5'-AGG GCT GTC AAT CAT GCT GG-3') and nts 3836-3855 (5'-AAA
CTC ACG GTC GGT GCA GC-3') of clone E4.1 of the human HD gene (Accession
number
L34020). PCR conditions used are described in Mangiarini et al.(1996). DNA was
extracted
from a tail clip and an ear punch from each mouse used in this study. Both
samples were
subjected to PCR genotype analysis. For in situ hybridization analysis, the
animals were
anesthetized with >100 mg/kg sodium pentobarbital, decapitated, the brains
removed and
stored at -70°C prior to sectioning. For RNA isolation, animals were
anesthetized,
decapitated and the striatum and cortex were excised and stored in liquid
nitrogen prior to
RNA extraction. Animal care was given according to protocols approved by
Dalhousie
University and the Canadian Council of Animal Care.
Differential display was used to identify novel mDNA or previously described
mDNA whose
relative expression levels are altered as a result of the presence of the
transgene. Using
differential display, the mRNA populations derived from the striatum of 10
week old wild type
were compared with age-matched R6/2 transgenic mice. Differential display has
been used
extensively (> 750 references) since its development (Lung and Pardee, 1992)
to identify
changes in gene expression in cells and in tissues including brain (Douglass
et al., 1995; Babity
et al., 1997a; Livesey et al., 1997; Berke et al., 1998). Perhaps the most
important finding
was the demonstration by Qu et al., ( 1996) that differential display can be
used to isolate
genes differentially expressed in inbred strains of mice. The power of
differential display is that
the sequence information obtained can be directly related to the experimental
paradigm.
Moreover, such sequence information includes sufficient information to
identify transcripts and
34
CA 02285690 1999-10-07
can then lead to experiments that reveal function of the cognate protein in
the experimental
model.
DNA sequence information of potentially differentially expressed cDNA can be
used to
S generate oligonucleotide probes for in situ hybridization to define the
anatomical and temporal
patterns of expression of specific transcripts (see Babity et al., 1997a).
This technique is
especially useful to study ch<~nges in steady-state levels of mRNA in
heterogeneous tissue such
as brain. Brain tissue can be micro-dissected (Babity et al., 1997b). This
enabled the present
inventors to reduce the requirement for tissue, and hence compare the mRNA
populations
derived from individual animals for each experimental group
Thus RT-PCR (Denovan-Wright et al., 1999) was used to identify differences in
the patterns
of gene expression between the striatum of wild-type and transgenic mice that
were
hemizygous for the 5' UTR, exon 1 and part of intron 1 of the human
Huntingon's Disease
gene. Total cellular RNA was isolated from the striatum and cortex of three 10
week-old
wild-type and three 10 week-old R6/2 HD mice (Mangiarini et al., 1996) and
used as the
template to generate single-stranded cDNA. Total cellular RNA from each animal
and tissue
was purified using TrizolT"" reagent (Gibco BRL) and the manufacture's
protocol. 10 pg
aliquots of total RNA were 'treated with RQ 1 DNAse-free DNAse (Promega) in
the presence
of DNAsinT"' (Promega) DNAse inhibitor to remove trace genomic DNA and then
converted
to single-stranded cDNA. The primers and conditions for PCR amplification
follow those of
the Deltas RNA fingerprynting manual (Clontech).
CA 02285690 1999-10-07
The eDNA was then used as the substrate for PCR reactions using 57
differential display
primer combinations. The radio-labelled PCR products were fractionated on a
denaturing
acrylamide sequencing gels using a Genomyx LRT"" sequencing apparatus,
transferred to 3MM
filter paper and dried. The dried acrylamide gels were exposed to
autoradiography film
(BioMax MRS) overnight. .After fractionating the radio-labelled PCR products
on denaturing
acrylamide gels, it was found that the overwhelming majority of the
approximately 18,000
PCR products screened were common to both the wild-type and HD mice (data not
shown).
One PCR product, amplified using the primers P7 (5'-ATT AAC CCT CAC TAA ATG
CTG
TAT G- 3') and T6 (5'- CA'T TAT GCT GAG TGA TAT CTT TTT TTT TCG- 3') of
approximately 500 bp, was observed in each of three samples derived from the
striatum of
wild-type mice (Fig. lA). This S00 by band was absent from the samples derived
from the
striatum of the HD mice (Fig. 1 A) and was absent from each of the samples
derived from the
cortical tissue (data not show).
Figure lA shows the Down-regulated in Huntington's Disease (DHD) transcript,
identified by
differential display RT PCR. A band of approximately 500 by (arrow) was
amplified from
cDNA made form 10 week-old wild-type but not 10 week-old HD striatal tissue.
Total RNA
from individual animals (numbered 1-6) was used as the substrate for the
generation of single-
stranded cDNA. Animals 1, 2 and 3 were transgenic HD mice. Animals 4, 5 and 6
were wild-
type mice.
36
CA 02285690 1999-10-07
EXAMPLE 2 - Cloning of I)HD
The 500 by band, designate DHDpcr, was excised from the dried gel and
rehydrated in 40 pl
of H20 for 10 min at room temperature. The eluted DNA was subjected to PCR re-
amplification using the P7 and T6 primers, rTaq polymerase (Pharmacia) and the
following
conditions: 60" @ 94°C, 19 :~c (30" @ 94°C, 30" @ 58°C,
120" @ 68°C + 4" per cycle), 7' @
68°C. The PCR reaction was subjected to agarose gel electrophoresis and
the 500 by band
was removed from the gel, extracted from the agarose using the Qiagen gel
extraction
protocol and cloned into the vector, pGem-T using standard methods. Plasmid
DNA was
isolated from selected transfi~rmants using Qiagen spin columns. 'The
resultant clone was
named pDHD.
EXAMPLE 3 - Identification of DHD
The cloned insert of pDHD was radio-labelled and used as a hybridization probe
in northern
blot analysis (Fig. 1B). Northern blots of total RNA were prepared using the
method
described in Denovan-Wrigl~t et al. (1998). The 500 by cloned insert of DHD
was radio-
labelled with [a-32P]dCTP (3000 Ci/mmol) using the Ready-to-Go dCTP beads
(Pharmacia).
Northern blot hybridization, brain tissue preparation and irr situ
hybridization are described in
Denovan-Wright et al. (1998). The S00 by cloned insert of pDHD annealed to a
transcript of
approximately 7.5 kb in total RNA isolated from the striatum of ten week-old
wild-type mice.
37
CA 02285690 1999-10-07
Figure 1B demonstrates that DHD is expressed in the striatum but not the
cortex of wild-type
mice and the steady-state levels of DHD are reduced in 10 week ald transgenic
HD mice. The
differential expression of DHD in HD mice was confirmed by northern blot
analysis. The
cloned insert of pDHD was radio-labelled and used as a hybridization probe in
northern blot
analysis. The northern blot was prepared by size-fractionating total RNA from
the striatum
and cortex of three individual 10 week-old HD ( 1, 2 and 3) and wild-type (4,
5 and 6) mice.
Following the hybridization of pDHD, the radio-label was removed and the blot
was
subsequently allowed to hybridize with a probe that detects constituitively
expressed
cyclophilin. The hybridization pattern of the cyclophilin probe is aligned
below the northern
blot demonstrating that equivalent amount of RNA were present in each lane.
The relative
mobility of RNA molecular weight standards (RNA ladder, Gibco BRL) are shown
on the left
of the northern blot.
The hybridization signal of pDHD was significantly lower in the RNA samples
derived from
the striatum of 10 week-old :EiD mice. No expression of the DHD mRNA was
detected in the
cortical RNA samples derived from either the wild-type or HD mice.
EXAMPLE 4 - Sequencing DHD
The sequence of the cloned dii~erential display band, pDHD, was determined
using M13
universal forward and reverse sequencing primers and the T7 sequencing kit
(Pharmacia). The
484 by cDNA fragment did not have sequence similarity to any Genbank entries.
38
CA 02285690 1999-10-07
Figure 2 shows the nucleotide sequence of the cloned DHD differential display
product,
pDHD. The position of the primers used to amplify the fragment are underlined
and labelled.
The nucleotide sequence and position of oligonucleotide probes 1 and 2 within
the pDHI~
sequence are shown.
EXAMPLE 5 - Localization of DHD
In order to identify the coding strand and to localize the transcript in the
wild-type mouse
brain, two oligonucleotide probes were designed (probe 1, 5'- GAA CAT GTA GCA
TAT
ACT CCA GAC AAC AGA TCA TAT GG - 3'; probe 2, 5' - CAG CTT CTC CAC AGG
AAC ACA GTA ACA AAG AG -3') that were complementary to different regions and
strands of the 484 by pDHD clone. These oligonucleotides were used for in situ
hybridization
analysis. Using high stringency post in situ hybridization washes (2 x 30' in
1X SSC @ 58oC,
4 x 15' in 1X SSC @ 58oC, 4 x 15' in O.SX SSC @ 58oC, 4 x 15' in 0.25X SSC @
58oC), it
was found that oligonucleotide probe 1 annealed with mltNA in the striatum,
nucleus
accumbens and olfactory tubercule of ten week-old wild-type mice (Fig. 3A).
The
hybridization signal was significantly reduced in the striatum, nucleus
accumbens and olfactory
tubercle of the 10 week-old I-ID mice (Fig. 3A).
Figure 3A shows in situ hybridization of probe 1 to coronal (top three
sections) and saggital
(bottom section) 10 week-old wild-type (WT) and HD mouse brain sections.
Specific
hybridization of the probe was observed in the striatum, nucleus accumbens and
olfactory
39
CA 02285690 1999-10-07
tubercle of wild-type mice. 'The top three sections represent the distribution
of DHD
throughout the rostral-caudal axis of the striatum.
The in situ hybridization results confirmed the northern blot analysis
demonstrating, 1 ) that the
expression of DHD mRNA was restricted to the striatum, nucleus accumbens and
olfactory
tubercle and 2) that the levels of DHI~ mRNA were decreased in I~ mice
compared to the
wild-type. The probe did nol: anneal with mRNA in any other brain nuclei. No
hybridization of
oligonucleotide probe 2 was observed in any region of the brain in wild-type
or HD mice (Fig.
3). Based on this hybridizatiion, the coding strand, complementary to probe l,
of pDHD was
defined.
EXAMPLE 6 - Characterization of DHD
The in situ hybridization using oligonucleotide probe 1 demonstrated that DHD
mRNA levels
1 S in the striatum , nucleus accumbens and olfactory tubercule were decreased
in ten week- old
HD mice. By ten weeks of age, the HD mice all showed motor symptoms including
resting
tremor and stereotypic involuntary movements. Moreover, these mice immediately
clasped
their feet together and curled into a tight ball when picked up by their
tails.
As the phenotypic signs are progressive over a number of weeks, the present
inventors
examined whether the DHD transcript was ever expressed in the striatum of the
HD mice or
whether the steady-state levels of the transcript diminished in the striatum
in a course that
parallelled the development of the motor disorders. Wild-type and HD mice were
sacrificed at
CA 02285690 1999-10-07
5, 7 and 8 weeks of age and their brains were prepared for in situ
hybridization analysis using
probe 1 (Fig. 3B).
Figure 3B shows the levels of DHD mRNA decrease in HD mice over the period of
time that
the HD mice develop abnormal movements and postures. In situ hybridization
analysis of
coronal and saggital sections of wild-type and HD mouse brain using
oligonucleotide probe 1
which is complementary to the coding strand of DHD. At 5 weeks of age, before
the
development of motor symptoms, the HD mice express the DHD transcript in the
same brain
nuclei and at the same relative levels as wild-type mice. The steady-state
level DHD decreases
in the striatum, nucleus accumbens and olfactory tubercle from 5 to 10 weeks
in the HD but
not wild-type mice. By 9 wf;eks of age, the HD mice have abnormal movement and
posture.
The numbers refer to the age; in weeks of the wild-type (WT) and Huntington's
(HD)
transgemc mice.
None of the mice at these ages had overt motor symptoms. Sections taken
throughout the
rostral-caudal axis of the striatum showed that DHD was expressed in the 5
week-old wild-
type and HD mice. The relative hybridization of probe 1 did not change in 5,
7, 8 and 10
week-old wild-type mice. The intensity of the hybridization signal appeared to
decrease in the
striatum, nucleus accumbens and olfactory tubercle of HD mice from S to 10
weeks compared
to their wild-type litter mates (Fig. 3B).
One day old wild-type and HD mice were frozen, sectioned on a cryostat and
whole mouse
sections were prepared for i~r situ hybridization using probe 1. The same high
stringency
41
CA 02285690 1999-10-07
post-hybridization washing conditions were employed for the one day-old mouse
body
sections as were used for the adult mouse brain sections. Parallel in situ
hyridization
experiments using the probe :2 were performed in order to determine the level
of non-specific
signal in the mouse sections. Probe 1 specifically annealed to the developing
striatum (Fig.
3C).
Figure 3C demonstrates that DHD is expressed in the developing striatum of one
day-old
wild-type and HD mice. The: sections on the left were subjected to in situ
hybridization using
probe 1. Following hybridization, the sections were counter-stained with
cresyl violet to
visualize the mouse organs. The signal outside the brain was non-specific as
probe 2 and other
unrelated control oligonucleotide probes all labelled these tissues.
There was no difference in the pattern of hybridization between the one day-
old wild-type and
HD mice demonstrating that DHD was expressed in the developing brain of both
wild-type
1 S and HD mice.
Following in situ hybridization, the sections were covered in autoradiographic
emulsion, left in
the dark to expose for 4 weeks and then developed and viewed under dark-field
microscopy
or, after counter-staining the sections with cresyl violet to visualize
neuronal cell bodies, under
bright-field microscopy. Silver grains were observed to be concentrated in the
striatum of the
wild-type mice. Figure 3D shows emulsion autoradiography of mouse brain
sections following
in situ hybridization of probE; 1 demonstrated that the DHD transcript is
expressed in neurons.
DHD is not homogeneously distributed throughout the mouse striatum. Dark field
illumination of the sections after emulsion autoradiography showed that the
silver grains were
42
CA 02285690 1999-10-07
clustered in specific regions of the 10 week old wild-type mouse striatum (A
and C). Sections
from 10 week old HD mice subjected to identical in situ and emulsion
autoradiographic
conditions are shown in B and D. The photomicrographs shown in A and B were
viewed
using the lOX objective (bar represents 100 Vim). The micrographs shown in C
and D, were
viewed under the 20X objective (bar represents 25 Vim). The insert in panel C
is a portion of
the section in A and C counter-stained with cresyl violet to visualize the
neurons, viewed using
the 40X objective under bright filed illumination. Note the distribution of
the silver grains
over some, but not all, of the: striatal neurons as well as being concentrated
around clusters of
neurons. It appeared that the silver grains were absent from fibre tracks
within the striatum.
It appeared that DHD mRNA was not confined to regions close to the nucleus but
was
dispersed in cellular processes.
EXAMPLE 7 - Confirmation of DHD in other Mammalian Species (Rat)
The oligonucleotide (probe 1. ) complementary to the coding strand of the DHD
transcript, was
also used as an in sitzs hybridization probe against coronal brain sections
derived from adult
rats. Figure 4 shows in situ hybridization analysis of adult rat brain
sections using
oligonucleotide probe 1 complementary to the coding-strand of DHI~ revealed
that the pattern
of expression of DHD is the same in rats and mice. The hybridization
conditions used to
detect the rat homologue of DHD in rat brain tissue differed from those used
to detect the
transcript in mice only in that the stringency of the post-hybridization
washes were reduced.
43
CA 02285690 1999-10-07
No hybridization was observed in the rat striatum using the post-hybridization
washes
employed following the in situ hybridization of mouse brain sections. However,
when the
stringency ofthe post-hybridization washes was lowered (2 x 60' in 1X SSC @
42oC, 2 x 60'
in O.SX SSC @ 42oC, 2 x 60' in 0.25X SSC @ room temperature), the DHD
oligonucleotide
probe specifically labelled the adult rat striatum, nucleus accumbens and
olfactory tubercule in
a pattern indistinguishable from that observed in mouse brain sections. It
appears, therefore,
that a transcript which shares nucleotide sequence and expression pattern is
present in both
mice and rats. The evolutionary conservation of DHD suggests that it is
important for normal
fiznction of the basal ganglia.
The mechanism by which only a small group of neurons in the striatum and
cortex are
rendered vulnerable to this ubiquitously expressed mutant protein is not
known. The present
inventors hypothesize that the presence of the expanded polyglutamine tract in
huntingtin
alters gene expression in the striatum.
EXAMPLE 8 - Analysis of :DHD in Genomic DNA
Genomic DNA was isolated from wild-type and HD mice and subjected to Southern
blot
analysis using pDHD as a hybridization probe. Analysis of the size of the
fragments that
hybridized with pDHD demonstrated that there was no difference in the size of
the hybridizing
fragments between the wild-type and HD mice. Figure 5 shows the genomic DNA
restriction
fragments that hybridized with pDHD were the same in wild-type and HD mice.
The size of
the hybridizing BamHI and EcoIRI fragment s in each genomic DNA sample is
approximately 8
kb and 3 kb, respectively. This Southern blot analysis indicates that the gene
encoding DHD
44
CA 02285690 1999-10-07
is present as a single-copy in the mouse genome. The numbers at the left of
the blot are the
relative mobility of molecular weight markers ( 1 kb ladder, BioRad).
The size of the hybridizing f~amHI and EcoRI fragments in each genomic DNA
sample is
approximately 7 kb and 3 kb, respectively. The fact that only one genomic DNA
fragment
hybridized with the DHD probe indicates that a single copy of the gene is
present in the mouse
genome.
EXAMPLE 9 - Isolation and Characterization of cDNA DHD
In order to isolate DHD cDNA clones, oligonucleotide probes 1 and 2 were used
in 5' and 3'
Rapid Amplification of cDNA Ends (RACE) reactions using commercially, prepared
RACE-
ready mouse striatal cDNA (Clontech). Several independent clones were isolated
and those
that contained the sequence of pDHD were selected for further analysis. Each
of the S' RACE
clones was identical in sequence over the length that the clones could be
aligned. The
difference in length between these clones is a result of termination of the
original reverse-
tra.nscriptase reaction at different positions along the mRNA. No difference
in size or
sequence was detected between several 3' RACE clones. The longest S' RACE
clone and one
3' RACE clone were completely sequenced using internal primers. The present
inventors
were able to isolate a very short clone that extended the 5' RACE clone using
an internal
primer (probe 3, 5'- CTA TTT CAC AAG AGA CTG ACC AGC CAA TAA ATC TC- 3')
The compiled sequence of the first DHD cDNA clone, named cDHD-1 is presented
in Fig. 6.
cDHD-1 is 3235 by in length. The restriction map of cDHD-1 is shown in Fig. 7.
CA 02285690 1999-10-07
The mRNA that hybridized with pDHD was approximately 7.5 kilobases in length.
In order to
obtain DHD cDNA clone that was larger than cDHD-1, the present inventors
screened a
mouse brain cDNA library.Several clones were identified that hybridized with
the pDHD
probe. The sequence of the largest of these cDNA clones, cDHD-2, was
determined. The
sequence (Fig. 8) was 5753 base pairs in length. The restriction map of cDHD-2
is shown in
Fig. 9.
cDHD-1 and cDHD-2 share sequence identity over 2095 bp. However, the 5' 1142
by of
cDHD-1 and the 5' 1689 by of cDHD-2 are unique to each clone. Clone cDHD-
2extends
1969 by in the 3' direction compared to cDHD-1. A schematic showing the
regions of
sequence identity and the unique sequences of cDHD-1 and -2 are shown in Fig.
10.
46
CA 02285690 1999-10-07
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48
CA 02285690 1999-10-07
SEQUENCE LISTING
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DENOVAN-WRIGHT, Eileen
NOVANEURON, INC.
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CA 02285690 1999-10-07
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taatcattta ttataactca gacaaggtgt aaataaattc ataattcaaa cagccagtat 3000
atatgcatat atgggtgtta cattgcaaaa atctctatct ttgttctatt cacatgctta 3060
aagaagtaag aaatcttttg tggatatgta attatacata taaagtatat atatatgtat 3120
gatacatgaa atatatttag aaatgttcat aattttaatg gatatt~~ttt ggtgtgaata 3180
attgaataca acatttttaa aatgaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaa 3236
<210> 2
<211> 5752
<212> DNA
<213> mouse
<400> 2
aagtgtaaat aaaataaaca tctaataaaa aaaattacat accatagagg aacaagataa 60
tttctgccca acttcatacc ctccagcgta tagtgttgag gtt:tggtctg ttgctgtgta 120
ttgtaatgta atgttaaatt ctctacctga aggtctaggc ctacaagtga attctcatgt 180
ttatagagtt ttgttgtgca aaccttgttc cttaatttaa aactatggtt aaaaaacaaa 240
acaaaactgg ctacagccaa taactgaagg gggttacctt gttgaagggg tggaaaagag 300
agaggaggaa gaagggagtt caagagaagg agaagaacaa gaggagagga ggaagctgcc 360
acgaggggag atgggccatg agaacttggc caggagaaat agccagtatc tggagtacac 420
cactgaggag gtagccaggc tagcagttag aagagtagat taggggttat ttttccccca 480
ctccacatag ttatcaaagc caaataaaat aaccatagtc tg<~gtctcat ctatttgtaa 540
gctagttggg tataagatta atttggctgt actacagttt agatttctaa cataggaact 600
atcaaaaact tgctcaaaca agaacatgct gacaatattt taaaatgatt atttatattg 660
tttgcacttt ctaaagtttc ttctaaatgt tccatggtca aattaaaaaa tatacatatt 720
2
CA 02285690 1999-10-07
ggctattaaa ttcgtctaag 'tggggctgga gagatagctc agaggttaag agcactgact 780
gctcttccag aggtcctgag ttcaattccc agcgaccaca tggtggctca cagccatctg 840
taatagatag gatctgacgc ~~ctcttctgg agtgtctgaa gacagctaca atgtactcat 900
atatattaaa taaataatat tagaaaattc ttctaagtgt atcatttata gaatatttaa 960
tatataaagt aaatgcctca ggaaatataa acttggaatt aaat:caaaga acttcatgag 1020
tagtgggcca caaaaaatgt gtaccagggg aagaccggag ggaggggaga aggaagggat 1080
ggagatagaa ttttgcctct ~3cattccttg ggctggcaca ggtataatgc tgtgggaatt 1140
gggaaactac aaggaagctg caaagctggg cggaactcgt ttcc:gcaagc tgggctcatc 1200
taagtgtcca tgcatggctg ccacactgca gtgaacttta aaac:atttgt gttccagaga 1260
tgtagagatg ctcacaatag tacaaaggcg ggagggaggt atttccagac taagaggaag 1320
aaaaaccatt gctgattaaa catctgcata tgagcgcccc cacca ccata cacacacaca 1380
cacacacaca cacacacaca caaccaaaca gaacaaatac acatgcatgt ctacagcctg 1440
caggaacaaa atggtatgtc tgtgaggaac caggagatgc acaggtccta acctctgtct 1500
cctacaagcc ctgaagtctg gtcagggtca aatgtacaaa agcaggctaa ggaagctgtt 1560
tagtgaaaga tttttttctt caactctagg aacaacctat ttcctaggat ttggagagtg 1620
ctcaggagga aacattcaga caactgatgc tctctgtgta ccc<~agattc aggtattggg 1680
gtagttagtt gtgctcatgt atgtgctaga tatattagca cagcctgcct tctgctgcac 1740
aacgccttag agacccggcc tttcaatgag cttagcttgt gctctgtttc tgctctctta 1800
ggtctaaact atggtgtcag ttttaataga acaaaagtat gcatcttgcc ttggcttgag 1860
ccttttcgtt ttcaatgctg acttctcccc tttctctcct gtgctcacct tacctttcca 1920
gagtgtaagg gacaactttt aaggaggcgt gtccctggta ggg~3catccc tgttcaccag 1980
gtgcctgtca tcaccccact tgactgacat ctaccctggt gactatgggt tcctcttgtt 2040
tgtagggaac ggtggctcca ggtggaggca t:caatctgtt gggttctggt tcccggctgc 2100
ctttggtttt gaaagtctct tctctgtata ttcctaccct gcatttgctt tgtgtggtgc 2160
tgatgctgtg cgcagcagga ttcttggatg actctccatc agtcacagac tccccctgtt 2220
gcaaagtgtc aggctgactc gacagtcacc gtaaaatctg agtcagtcac acacaggctg 2280
tcagccacgg cttccacttg catggctatt ctattttcac acgtgagttt ctgttgctgg 2340
ctggctgact ggcattatct atgctaagtt gaaatcaggg gtgcccagca gagcccatca 2400
ttctcactgt ctttgaaaca aagctgtacg gtttgatcga tgaacgt.att taaagcattt 2460
catgcaatga caaagtgctc agtagtggaa ggcaggctgt gaccagtctg cctgctcctt 2520
actataattg tgaggatttg ttactggaac agtacatgga ggcctgacct tgtgggggca 2580
cagggtggaa ccttagctga atatagtgtg tgtctcaaga ggaagtcagg gtactagctc 2640
agtgctcaat ctccaggtac tatatataca tttgcccgtt ttatctctaa tgtgaaataa 2700
atccccaaac acttgtttat cgtgtagcgt acctaaaaga ctattct:att atgggtgtcc 2760
ccactttctt ggtttggtca ccccgatccc ccggtcttct gctgtatcta gaacagtgac 2820
tataaatgat gtatgggaat agtgtttcca tatgatctgt tgtctggagt atatgctaca 2880
tgttcattta ctgtacaaaa acccagtgca gctgatgatg caaagcagtc tctctctgtg 2940
tacagtgccc -cacctattta aaaatcacgt acttgcccag aacactqtga aacacttaac 3000
ataagaacaa acgcagcgtc tggattcttt ccaaggagag cagcttt:ctc cacaggaaca 3060
cagtaacaaa agaggtccgc cgccatccac acccagccaa gacacct:cag aggccatagg 3120
gacaacctcc ttgctggcca acacctgctg gagcaggggc acaggt<:cca gcaactgatc 3180
ctcagtggat gggtctgcag ccaaagcctt aatgggctct cttttgaagg ggaaagaaag 3240
aatttcaagc ttatgatatc caatattatt atagttgatg agttagt:aaa ttccaaaaaa 3300
aaaagatgat tttatatgta tgacataaaa aaaatctttg taaagtgcgc aagtgcaata 3360
atttaaagag gtcttatctt tgcatttata aattataaat attgta<:atg tgtgtaattt 3420
ttcatgtatt catttgcagt ctttgtattt aaaaaaactt tactgttatg tttgtataat 3480
agaacattaa tcatttatta taactcagac aaggtgtaaa taaattcata attcaaacag 3540
ccagtatata tgcatatatg ggtgttacat tgcaaaaatc tct.atctttg ttctattcac 3600
3
CA 02285690 1999-10-07
atgcttaaag aagtaagaaa tcttttgtgg atatgtaatt atacatataa agtatatata 3660
tatgtatgat acatgaaata tatttagaaa tgttcataat tttaatggat attctttggt 3720
gtgaataatt gaatacaaca tttttaaaat aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 3780
aaaatttttt tttttttttt ~~tattccaga gattaaagac actagatctt taaccttgaa 3840
gggcaggcaa gaggtcggca ~3tgctgtcaa catagaagtc agggaccatt ttcttcttga 3900
acatgcagtc actttcctga ttgctcttca catcctcaag gctccggaat tccgggggtg 3960
tggtgggctt tgatctcagg <3ctctggagg cagaagcagg cagatctctg tgaatatgag 4020
gccagcctgc actacacaga gctccagacc agtcatggct acat:catgaa accctgtctc 4080
aaaaagaaaa taaaaactgt t:gtgtttcta ccatagtgtt aaactcagag tctgagtaat 4140
gtcgggctga catgctcggg t:gtttaacat accttcagct ttgacgaggc gctgaacagt 4200
caaagtctgg ccttggggag c:ggtggctgt gtttgtgctc aagtccaccg tgaaatcctg 4260
attgtgaatt tggacaaccg t:gtccttctt cttggccttc catgcaacct ccaacttcat 4320
gttggtcatt ttgtcaaaac actgtgtgat gtttttatca atatactgcc attccacata 4380
tgtagagatg tagtctgcct dgctttcctt ttctttagcc aatcgaatgc tcttgatcat 4440
gccctcaatc tcatctctag catttatcac gtctctgcta attcctgaaa cttgaatcga 4500
agttttcttc tggttcatct c:aatggtgat gttcagttcc ttctgaatct cattcagttt 4560
ctcgtactcc tccatgtcaa agtcactgac acactcatcg tcattggtgt aggaaagctg 4620
ctctttggta atcagttcct t:tagccagga gattgttttg ttcacactgt ctacccctga 4680
accacatacc tggaaaactg t:gtgctctat tttcttttcc aaaaccaggg tgttcttttt 4740
gggggaagct tgcttgggaa agccaagaaa ggctaaagag aaaatggaaa ttaatgtttc 4800
ttttactccc ttcaacatca aggttaggaa tatgtatttc ataaaag<aa acaactcaca 4860
ggcaatctta gacatcactg actgcttggc aggcgactgc ttggggggag ctggagagcc 4920
ttctcttt.ct ttcatgttgt c:gtaaaaaaa ttgcagaata tggggctgga agataacaac 4980
tttaactctc ttcacagcct c~cactgattt tttctggaca aattcttcaa tggcatctat 5040
tatcgctttt gctactacgt t.tgggtcctg ttgagcattt ccttcaaaaa caaaaaaagc 5100
acatttttaa aaagtcaagg t.taagatcca cctgcaaaaa aaagctgcaa tataagcgag 5160
gaattctagt tgtcacagga a.ataaaaatg tctgttccca ctataatcaa tgtagactga 5220
taatattatg ccagcaaata a~ttttgaagt cctaggcaca gtgggaggag gttttgttcc 5280
acgctgttca taagccaata ccccagcaaa agaccttaaa ggacaacttg taatttggga 5340
cattcacatc tgtcctcttc atctgatctg gctcccagtg tcactctca a acacggtcct 5400
tagagggaca atttatccct gcctctgctt gatcttatgc atgtatctgt attcttccag 5460
ccatccctgg cgacctgatt tttctaaggc acccaaaact gtaagctact tcttataatc 5520
tataattctg agcatattag ttagcctgag cctccaggat atctttcttc cctatactca 5580
gtccagtttt agctgcccag aaggattcaa agctgatcta cgagtagatc actcctgtct 5640
acagcttgtt ccagatcttg tttctcaagc cctggaagcc atcagccagg taagattgta 5700
aaacaatccc tttctaatca tgggtgtggc ccaaagtgaa tggccggaat tc 5752
<210> 3
<211> 475
<212> DNA
<213> mouse
<400> 3
tgtatgggaa tagtgtttcc atatgatctg ttgtctggag tatatgctac atgttcattt 60
actgtacaaa aacccagtgc agctgatgat gcaaagcagt ctctctctgt gtacagtgcc 120
ccacctattt aaaaatcacg tacttgccca gaacactgtg aaacacttaa cataagaaca 180
aacgcagcgt ctggattctt tccaaggaga gcagctttct ccacaggaac acagtaacaa 240
aagaggtccg ccgccatcca cacccagcca agacacctca gaggccatag ggacaacctc 300
4
CA 02285690 1999-10-07
cttgctggcc aacacctgct ggagcagggg cacaggtccc agcaactgat cctcagtgga 360
tgggtctgca gccaaagcct taatgggctc tcttttgaag gggaaagaaa gaatttcaag 420
cttatgatat ccaatattat tatagttgat gagttagtaa attccaaaaa aaaaa 475
<210> 4
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of P,rtificial Sequence: primer
<400> 4
agggctgtca atcatgctgg 20
<210> 5
<211> 20
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: primer
<400> 5
aaactcacgg tcggtgcagc 20
<210> 6
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: probe
<400> 6
attaaccctc actaaatgct gtat 24
<210> 7
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: probe
<400> 7
cattatgctg agtgatatct ttttttttcg 30
CA 02285690 1999-10-07
<210> 8
<211> 38
<212> DNA,
<213> Artificial Sequence
<220>
<223> Description of F.rtificial Sequence: probe
<400> 8
gaacatgtag catatactcc agacaacaga tcatatgg 3g
<210> 9
<211> 32
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: probe
<400> 9
cagcttctcc acaggaacac agtaacaaag ag 32
<210> 10
<211> 35
<212> DNA
<213> Artificial Sequence
<220>
<223> Description of Artificial Sequence: primer
<400> 10
ctatttcaca agagactgac cagccaataa atctc 35
6
