Note: Descriptions are shown in the official language in which they were submitted.
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GENES ASSOCIATED WITH NEUROTRANSMITTER PROCESSING
A portion of the disclosure of this patent document contains material which is
subject to
copyright protection. The copyright owner has no objection to the facsimile
reproduction by
anyone of the patent document or the patent disclosure, as it appears in the
Patent and Trademark
Office patent f le or records, but otherwise reserves all copyright rights
whatsoever.
TECHNICAL FIELD
The invention relates to five genes associated with neurotransmitter
processing and their
corresponding gene products. The invention also relates to the use of these
biomolecules in
diagnosis, treatment, prevention, and evaluation oftherapies for neurological
and hormone-related
diseases, more particularly Parkinson's disease, schizophrenia, and epilepsy.
BACKGROUND OF THE INVENTION
Normal neurological function requires a careful balance of neurotransmitters.
An upset in
the balance can lead to disease. For example, Parkinson's disease is triggered
by an as yet
unknown event that increases the susceptibility of dopaminergic neurons to
oxidative processes
which in turn leads to cell damage and death (Jenner, P. ( 1998) Mov. Disord.
13:24-34). The
resulting lack of dopamine leads to tremors and rigidity in the patient, the
main clinical
manifestations of Parkinson's disease (Birkmayer, W. and P. Riederer ( 1986)
Understanding_the
Neurotranmitters: Kevs to the Workin~~s of the Brain, Springer-Verlag, New
York NY).
Many other disorders result from an improper balance among neurotransmitters.
The
central dopaminergic neurons have long been thought to play a significant role
in schizophrenia
(Farde, L. (1997) Schizophr. Res. 28:157-162), but a more complex picture is
emerging where
interactions among dopamine, norepinephrine and other neurotransmitters
appears to be the basis
of the disorder (Carlsson, A. et al. ( I 997) Life Sci. 61:75-94). Both
dopamine and norepinpehrine
are also implicated in attention deficit hyperactive disorder (Hechtman, L.
(1994) J. Psychiary
Neurosci. 19: 193-201 ). In animal models norepinephrine has been implicated
in epilepsy (Szot,
P. et al. ( 1996) Brain Res. Mol. Brain Res. 43:233-245; Clough, R. W, et al.
( 1998) Epilepsy Res.
29:135-146); Janumpalli, S. et al. (1998) J. Neurosci. 18:2004-2008).
Norepinephrine directly
regulates gonadotropin-releasing hormone and may therefore be involved in
reproductive
disorders (Becu-Villalobos, D. et al. ( 1997) Cell Mol. Neurobiol. 17:699-715;
Pau, K.Y. and H.G.
Spies (1997) Chin. J. Physiol. 40:181-196). Norepinephrine also regulates
growth hormone and
inhibition of dopamine /3 hydroxylase {DBH). DBH retards growth in animal
models
(Malozowski, S. et al. (1993) Acta Endocrinol. 129:554-558).
In general, neurological disorders are treated with drugs that modulate
neurotransmitter
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level in an attempt to maintain the correct level. Consequently, any of the
substrates, enzymes,
receptors or neurotransmitter transporters as well as genes involved in their
regulation are
potentially of therapeutic use. We have discovered five new genes that exhibit
significant
coexpression with genes known to be involved in the synthesis and release of
the
neurotransmitters dopamine, norepinephrine and epinephrine. These new genes
are useful in the
diagnosis, prevention, treatment, and evaluation of therapies for diseases,
particutarly neurological
and hormone-related diseases, more particularly Parkinson's disease,
schizophrenia, and epilepsy.
SUMMARY OF THE INVENTION
In one aspect, the invention provides for a substantially purified
polynucleotide
comprising a gene that is coexpressed with. one or more neurotransmitter-
processing-specific
genes in a plurality of biological samples. Preferably, each neurotransmitter-
processing-specific
gene is selected from(a) a polynucleotide sequence selected from the group
consisting of SEQ ID
NOs: 1-5; (b) a polynucleotide sequence which encodes the polypeptide sequence
of SEQ ID NO:
6; (c) a polynucleotide sequence which is complementary to the poiynucleotide
sequence of (a)
or (b); and (d) a probe which hybridizes to the polynucleotide of (a), (b), or
(c). Furthermore, the
invention provides an expression vector comprising any of the above described
polynucleotides
and host cells comprising the expression vector.
In a second aspect, the invention provides a substantially purified
polypeptide comprising
the gene product of a gene that is coexpressed with one or more
neurotransmitter-processing-
specific genes in a plurality of biological samples. The neurotransmitter-
processing-specific gene
may be selected from the group consisting of L-tyrosine hydroxylase (TH),
aromatic amino acid
decarboxylase (AADC), dopamine (i-hydroxylase (DBH), nicotinic acetylcholine
receptor a3
subunit precursor (nAchR-a3), secretogranin I and II, Rab3a, human cocaine and
amphetamine
regulated transcript (hCART), vesicular monoamine transporter 1 (hVMATI ), and
ARIX
homeodomain protein. Preferred embodiments are (a) the polypeptide sequence of
SEQ ID NO: 6,
and (b) a polypeptide sequence comprising at least 6 sequential amino acids of
the polypeptide
sequence of (a) . Additionally, the invention provides antibodies that bind
specifically to any of
the above described polypeptides.
In another aspect, the invention provides a pharmaceutical composition
comprising the
polynucleotide or the polypeptide of the inventions in conjunction with a
suitable pharmaceutical
carrier and a method for treating or preventing disease comprising
administering to a subject in
need such a composition in an amount effective for treating or preventing said
disease.
In yet a further aspect, the invention provides a method for diagnosing a
disease or
condition associated with the altered expression of a gene that is coexpressed
with one or more
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neurotransmitter-processing-specific genes in a sample, wherein each
neurotransmitter-
processing-specific gene is selected from the group consisting of L-tyrosine
hydroxylase (TH),
aromatic amino acid decarboxylase (AADC), dopamine (3-hydroxylase (DBH),
nicotinic
acetylcholine receptor a3 subunit precursor (nAchR-a3), secretogranin 1 and
II, Rab3a, human
cocaine and amphetamine regulated transcript (hCART), vesicular monoamine
transporter 1
(hVMATI ), and ARIX homeodomain protein. The method comprises providing the
sample
comprising one of more of said coexpressed genes; hybridizing the
polynucleotide to said
coexpressed genes under conditions effective to form one or more hybridization
complexes; and
detecting the hybridization complexes, wherein the presence of one or more of
the hybridization
complexes correlates with the presence of the disease or condition.
BRIEF DESCRIPTION OF THE SEQUENCE LISTING
The Sequence Listing provides exemplary gene sequences associated with
neurotransmitter processing, SEQ ID NOs: 1-5, and a polypeptide sequence, SEQ
ID NO: 6,
derived from the gene sequence of SEQ ID N0:4. Each sequence is identified by
a numerical
sequence identification number and by the Incyte clone number from which the
sequence was first
identified.
DESCRIPTION OF THE INVENTION
It must be noted that as used herein and in the appended claims, the singular
forms "a,"
"an," and "the" include the plural reference unless the context clearly
dictates otherwise. Thus,
for example, a reference to "a host cell" includes a plurality of such host
cells, and a reference to
"an antibody" is a reference to one or more antibodies and equivalents thereof
known to those
skilled in the art; and so forth.
DEFINIT10NS
"NSEQ" refers generally to a polynucleotide sequence of the present invention,
including
SEQ ID NOs: 1-S. "PSEQ" refers generally to a polypeptide sequence of the
present invention,
including SEQ 1D NO: 6.
A " variant" refers to either a polynucleotide or a polypeptide whose sequence
diverges
from SEQ ID NOs: 1-5 or SEQ ID NO: 6, respectively. Polynucleotide sequence
divergence may
result from mutational changes such as deletions, additions, and substitutions
of one or more
nucleotides; it may also occur because of differences in codon usage. Each of
these types of
changes may occur alone, or in combination, one or more times in a given
sequence. Polypeptide
variants include sequences that possess at least one structural or functional
characteristic of SEQ
1D NO: 6.
A "fragment" can refer to a nucleic acid sequence that is preferably at least
20 nucleic
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acids in iength, more preferably 40 nucleic acids, and most preferably 60
nucleic acids in length,
and encompasses, for example, fragments consisting of nucleic acids 1-SO of
SEQ ID NOs: l-5. A
"fragment" can also refer to polypeptide sequences which are preferably at
least 5 to about 15
amino acids in length, most preferably at least 10 amino acids long, and which
retain some
biological activity or immunological activity of, for example, SEQ ID N0:6.
"Neurotransmitter-processing-specific genes" refers to genes which are
involved in the
processing, such as the synthesis or release, of neurotransmitters. For
purposes of this invention,
the neurotransmitters are preferably dopamine, norepinephrine and epinephrine.
"Gene associated with neurotransmitter processing" refers to a new gene
sequence whose
expression pattern is similar to that of the neurotransmitter-processing-
specific genes and which is
useful in the~diagnosis, treatment, or prevention of diseases associated with
the altered expression
of neurotransmitter processing genes. The gene sequences can also be used in
the evaluation of
therapies for these diseases.
"Substantially purified" refers to a nucleic acid or an amino acid sequence
that is removed
from its natural environment and is isolated or separated, and is at least
about 60% free, preferably
about 75% free, and most preferably about 90% free from other components with
which it is
naturally present.
THE INVENTION
The present invention encompasses five genes useful in the diagnosis,
treatment,
prevention, and evaluation of therapies for diseases related to improper
neurotransmitter
processing, particularly neurological and growth-related diseases, more
particularly Parkinson's
disease, schizophrenia, and epilepsy. These genes were identified using the
method disclosed in
the US patent application entitled "Prostate Cancer-Associated Genes", Walker
et al., Serial
No:09/102,615 filed June 22, 1998, herein incorporated by reference, and
described briefly below.
The method provides first identifying polynucleotides that are expressed in a
plurality of
cDNA libraries. The polynucleotides may include genes of known function, genes
known to be
specifically expressed in a specific disease process, subcellular compartment,
cell type, tissue type,
or species. Additionally, the polynucleotides include genes of unknown
function. The expression
patterns of the known genes are then compared with those of the genes of
unknown function to
determine whether a specified coexpression probability threshold is met.
Through this
comparison, a subset of the polynucleotides having a high coexpression
probability with the
known genes can be identified. The high coexpression probability correlates
with a particular
coexpression probability threshold which is less than 0.001, and more
preferably less than
0.00001.
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The polynucleotides may originate from cDNA libraries derived from a variety
of sources
including, but not limited to, eukaryotes such as human, mouse, rat, dog,
monkey, plant, and yeast
and prokaryotes such as bacteria and viruses. These polynucleotides can also
be selected from a
variety of sequence types including, but not limited to, expressed sequence
tags (ESTs), assembled
polynucleotide sequences, full length gene coding regions, introns, regulatory
sequences, 5'
untranslated regions, and 3' untranslated regions. To have statistically
significant analytical
results, the polynucleotides need to be expressed in at least three cDNA
libraries. In one preferred
embodiment, the polynucleotides are obtained from sequence databases, such as
the L1FESEQ
database (Incyte Pharmaceuticals, Palo Alto CA).
The cDNA libraries used in the coexpression analysis of the present invention
may be
obtained from blood vessels, heart, blood cells, cultured cells, connective
tissue, epithelium, islets
of Langerhans, neurons, phagocytes, biliary tract, esophagus, gastrointestinal
system, liver,
pancreas, fetus, placenta, chromaffin system, endocrine glands, ovary, uterus,
penis, prostate,
seminal vesicles, testis, bone marrow, immune system, cartilage, muscles,
skeleton, central
nervous system, ganglia, neuroglia, neurosecretory system, peripheral nervous
system, bronchus,
larynx, lung, nose, pleurus, ear, eye, mouth, pharynx, exocrine glands,
bladder, kidney, ureter, and
the like. The number of cDNA libraries selected can range from as few as 20 to
greater than
10,000. Preferably, the number of the cDNA libraries is greater than 500.
In a preferred embodiment, gene sequences are assembled to reflect related
sequences,
such as assembled sequence fragments derived from a single transcript.
Assembly of the
polynucleotide sequences can be performed using sequences of various types
including, but not
limited to, ESTs, extensions, or shotgun sequences. In a most preferred
embodiment, the
polynucleotide sequences are derived from human sequences that have been
assembled using the
algorithm disclosed in the US provisional patent application entitled
"Database and System for
Storing, Comparing and Displaying Related Biomolecular Sequence Information",
Lincoln et al.,
Serial No:60/079,469, filed March 26, 1998, herein incorporated by reference.
Experimentally, differential expression of the polynucleotides can be
evaluated by
methods including, but not limited to, differential display by spatial
immobilization or by gel
electrophoresis, genome mismatch scanning, representational difference
analysis, and transcript
imaging. Additionally, differential expression can be assessed by microarray
technology. These
methods may be used alone or in combination.
Neurotransmitter-processing-specific genes can be selected based on the
current use of
these genes as diagnostic markers or as therapeutic targets for diseases
related to incorrect
neurotransmitter processing, such as the neurotransmitter processing. Genes
related to
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neurotransmitter processing include L-tyrosine hydroxylase {TH), aromatic
amino acid
decarboxylase (AADC), dopamine ~i-hydroxylase (DBH), nicotinic acetylcholine
receptor a3
subunit precursor (nAchR-a3), secretogranin I and 1I, Rab3a, human cocaine and
amphetamine
regulated transcript (hCART), vesicular monoamine transporter 1 (hVMATI), ARIX
homeodomain protein, and the like.
The procedure for identifying new genes that exhibit a statistically
significant
coexpression pattern with known genes related to neurotransmitter processing
is as follows. First,
the presence or absence of a gene sequence in a cDNA library is defined: a
gene is present in a
cDNA library when at least one cDNA fragment corresponding to that gene is
detected in a cDNA
sample taken from the library, and a gene is absent from a library when no
corresponding cDNA
fragment is detected in the sample.
Second, the significance of gene coexpression is evaluated using a probability
method to
measure a due-to-chance probability of the coexpression. The probability
method can be the
Fisher exact test, the chi-squared test, or the kappa test. These tests and
examples of their
applications are well known in the art and can be found in standard statistics
texts {Agresti, A.
(1990 Categorical Data Analysis. Wiley, New York NY; Rice, J. A. {1988)
Mathematical Statistics
and Data Analysis. Wadsworth & Brooks/Cole, Pacific Grove CA). A Bonferroni
correction
(Rice, supra, page 384) can also be applied in combination with one of the
probability methods for
correcting statistical results of one gene versus multiple other genes. In a
preferred embodiment,
the due-to-chance probability is measured by a Fisher exact test, and the
threshold of the due-to-
chance probability is set to less than 0.001, more preferably less than
0.00001.
To determine whether two genes, A and B, have similar coexpression patterns,
occurrence
data vectors can be generated as illustrated in Table 1, wherein a gene's
presence is indicated by a
one and its absence by a zero. A zero indicates that the gene did not occur in
the library, and a
one indicates that it occurred at least once.
Table I . Occurrence data for genes A and B
Library Library Library ... Library
1 2 3 N
gene 1 1 0 ... 0
A
gene 1 0 1 0
B
...
For a given pair of genes, the occurrence data in Table 1 can be summarized in
a 2x2 contingency
table.
Table 2. Contingency table for co-occurrences of genes A and B
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Gene A present Gene A absent Total
Gene B present 8 2 10
Gene B absent 2 18 20
Total 10 20
30
Table 2 presents coexpression data for gene A and gene B in a total of 30
libraries. Gene
A and gene B each occurs 10 times in the libraries. Table 2 summarizes and
presents 1 ) the
number of times gene A and B are both present in a library, 2) the number of
times gene A and B
are both absent in a library, 3) the number of times gene A is present while
gene B is absent, and
l0 4) the number of times gene B is present while gene A is absent. The upper
left entry is the
number of times the two genes co-occur in a library, and the middle right
entry is the number of
times neither gene occurs in a library. The off diagonal entries are the
number of times one gene
occurs while the other does not. Gene A and B are both present eight times and
absent 18 times,
gene A is present while gene B is absent two times, and gene B is present
while gene A is absent
two times. The probability ("p-value") that the above association occurs due
to chance as
calculated using a Fisher Exact test is 0.0003. Associations are generally
considered significant if
a p-value is less than 0.01 (Agresti, supra; Rice, supra).
This method of estimating the probability for coexpression of two genes makes
several
assumptions. The method assumes that the libraries are independent and are
identically sampled.
However, in practical situations, the selected cDNA libraries are not entirely
independent because
more than one library may be obtained from a single patient or tissue, and
they are not entirely
identically sampled because different numbers of cDNA's may be sequenced from
each library
(typically ranging from 5,000 to 10,000 cDNAs per library). In addition,
because a Fisher exact
coexpression probability is calculated for each gene versus 41,419 other
genes, the probability is
corrected for the multiple statistical tests by requiring a more stringent p-
value than the standard p-
value of 0.01.
Using the coexpression analysis method, we have identified five new genes that
exhibit
strong association, or coexpression, with known genes related to
neurotransmitter processing. The
results presented in Tables 5 to 10 show that the expression of the five new
genes have direct or
indirect association with the expression of known genes related to
neurotransmitter processing.
Therefore, the new genes can potentially be used in diagnosis, treatment, or
prevention of diseases
related to neurotransmitter processing, such as Parkinson's disease,
schizophrenia, epilepsy,
female reproductive disorders, attention deficit disorder, or in the
evaluation of therapies for these
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diseases. Further, the gene products of the five new genes are potential
therapeutic proteins and
targets of therapeutics against these diseases.
Therefore, in one embodiment, the present invention encompasses a
polynucleotide
sequence comprising the sequence of SEQ ID NOs: 1-5. These five
polynucleotides are shown by
the method of the present invention to have strong coexpression association
with known genes
related to neurotransmitter processing. The invention also encompasses a
variant of the
polynucleotide sequence, its complement, or 18 consecutive nucleotides of the
sequences provided
in the above described sequences. Variant polynucteotide sequences typically
have at least about
70%, more preferably at least about 85%, and most preferably at least about
9S% polynucleotide
sequence identity to NSEQ.
One preferred method for identifying variants provides using NSEQ and/or PSEQ
sequences to search against the GenBank primate (pri), rodent (rod), and
mammalian (mam),
vertebrate (vrtp), and eukaryote (eukp) databases, SwissProt, BLOCKS (Bairoch,
A. et al. (1997)
Nucleic Acids Res. 25:217-221 ), PFAM, and other databases that contain
previously identified
and annotated motifs, sequences, and gene functions. Methods that search for
primary sequence
patterns with secondary structure gap penalties (Smith, T. et al. (1992)
protein Engineering 5:35-
51) as well as algorithms such as BLAST (Basic Local Alignment Search Tool;
Altschul, S.F.
(1993) J. Mol. Evol 36:290-300; and Altschul et al. (1990) J. Mol. Biol.
215:403-410), BLOCKS
(Henikoff S. and Henikoff G.J. (1991) Nucleic Acids Research 19:6565-6572),
Hidden Markov
Models (HMM; Eddy, S.R. (1996) Cur. Opin. Str. Biol. 6:361-365; and
Sonnhammer, E.L.L. et al.
(1997) Proteins 28:405-420), and the like, can be used to manipulate and
analyze nucleotide and
amino acid sequences. These databases, algorithms and other methods are well
known in the art
and are described in Ausubel, F.M. et al. ( 1997; Short Protocols in Molecular
Biology°y, John
Wiley & Sons, New York NY ) and in Meyers, R.A. (1995; Molecular Bioloey and
Biotechnolo~v, Wiley VCH, Inc, New York NY, p 856-853).
Also encompassed by the invention are polynucleotide sequences that are
capable of
hybridizing to SEQ ID NOs: 1-5, and fragments thereof under stringent
conditions. Stringent
conditions can be defined by salt concentration, temperature, and other
chemicals and conditions
well known in the art.
For example, stringent salt concentration will ordinarily be less than about
750 mM NaCI
and 75 mM trisodium citrate, preferably less than about 500 mM NaCI and 50 mM
trisodium
citrate, and most preferably less than about 250 mM NaCI and 25 mM trisodium
citrate. Stringent
temperature conditions will ordinarily include temperatures of at least about
30°C, more
preferably of at least about 37°C, and most preferably of at least
about 42°C. Varying additional
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parameters, such as hybridization time, the concentration of detergent (sodium
dodecyl sulfate,
SDS) or solvent (formamide), and the inclusion or exclusion of carrier DNA,
are well known to
those skilled in the art. Additional variations on these conditions will be
readily apparent to those
skilled in the art (Wahl, G.M. and S.L. Berger (1987) Methods Enzymol. 152:399-
407; Kimmel,
A.R. (1987) Methods Enzymol. 152:507-511; Ausubel, supra; and Sambrook, J. et
al. {1989)
Molecular Cloning A Laboratory Manual, Cold Spring Harbor Press, Plainview,
NY).
NSEQ or the poiynucleotide sequences encoding PSEQ can be extended utilizing a
partial
nucleotide sequence and employing various PCR-based methods known in the art
to obtain full
length sequences or to detect upstream sequences, such as promoters and
regulatory elements.
(See, e.g., Dieffenbach, C.W. and G.S. Dveksler (1995; PCR Primer a Laboratory
Manual, Cold
Spring Harbor Press, Plainview, NY, pp. i-S; Sarkar, G. ( 1993; PCR Methods
Applic. 2:318-322);
Trigiia, T. et al. {1988; Nucleic Acids Res. 16:8186); Lagerstrom, M. et al.
(1991; PCR Methods
Applic. 1:111-119); and Parker, J.D. et al. (1991; Nucleic Acids Res. 19:3055-
306). Additionally,
one may use PCR and nested primers to walk genomic DNA. This procedure avoids
the need to
screen libraries and is useful in finding intron/exon junctions. For all PCR-
based methods,
primers may be designed using commercially available software, such as OLIGO
4.06 software
(National Biosciences, Plymouth MN) or another appropriate program, to be
about 18 to 30
nucleotides in length, to have a GC content of about 50% or more, and to
anneal to the template at
temperatures of about 68°C to 72°C.
In another aspect of the invention, NSEQ or the polynucleotide sequences
encoding PSEQ
can be cloned in recombinant DNA molecules that direct expression of PSEQ or
the polypeptides
encoded by NSEQ, or structural or functional fragments thereof, in appropriate
host cells. Due to
the inherent degeneracy of the genetic code, other DNA sequences which encode
substantially the
same or a functionally equivalent amino acid sequence may be produced and used
to express the
polypeptides of PSEQ or the polypeptides encoded by NSEQ. The nucleotide
sequences of the
present invention can be engineered using methods generally known in the art
in order to alter the
nucletide sequences for a variety of purposes including, but not limited to,
modification of the
cloning, processing, and/or expression of the gene product. DNA shuffling by
random
fragmentation and PCR reassembly of gene fragments and synthetic
oligonucleotides may be used
to engineer the nucleotide sequences. For example, oligonucleotide-mediated
site-directed
mutagenesis may be used to introduce mutations that create new restriction
sites, alter
glycosylation patterns, change codon preference, produce splice variants, and
so forth.
In order to express a biologically active polypeptide encoded by NSEQ, NSEQ or
the
polynucleotide sequences encoding PSEQ, or derivatives thereof, may be
inserted into an
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appropriate expression vector, i.e., a vector which contains the necessary
elements for
transcriptional and translational control of the inserted coding sequence in a
suitable host. These
elements include regulatory sequences, such as enhancers, constitutive and
inducible promoters,
and 5' and 3' untranslated regions in the vector and in NSEQ or polynucleotide
sequences
encoding PSEQ. Methods which are well known to those skilled in the art may be
used to
construct expression vectors containing NSEQ or polynucleotide sequences
encoding PSEQ and
appropriate transcriptional and translational control elements. These methods
include in vitro
recombinant DNA techniques, synthetic techniques, and in vivo genetic
recombination. (See, e.g.,
Sambrook, supra, and Ausubel, su ra).
A variety of expression vector/host cell systems may be utilized to contain
and express
NSEQ or polynucleotide sequences encoding PSEQ. These include, but are not
limited to,
microorganisms such as bacteria transformed with recombinant bacteriophage,
plasmid, or cosmid
DNA expression vectors; yeast transformed with yeast expression vectors;
insect cell systems
infected with viral expression vectors (baculovirus); plant cell systems
transformed with viral
expression vectors, cauliflower mosaic virus (CaMV) or tobacco mosaic virus
(TMV), or with
bacterial expression vectors (Ti or pBR322 plasmids); or animal cell systems.
The invention is not
limited by the host cell employed. For long term production of recombinant
proteins in
mammalian systems, stable expression of a polypeptide encoded by NSEQ in cell
lines is
preferred. For example, NSEQ or sequences encoding PSEQ can be transformed
into cell lines
using expression vectors which may contain viral origins of replication and/or
endogenous
expression elements and a selectable marker gene on the same or on a separate
vector.
In general, host cells that contain NSEQ and that express PSEQ may be
identified by a
variety of procedures known to those of skill in the art. These procedures
include, but are not
limited to, DNA-DNA or DNA-RNA hybridizations, PCR amplification, and protein
bioassay or
immunoassay techniques which include membrane, solution, or chip based
technologies for the
detection and/or quantification of nucleic acid or protein sequences.
Immunological methods for
detecting and measuring the expression of PSEQ using either specific
polyclonal or monoclonal
antibodies are known in the art. Examples of such techniques include enzyme-
linked
immunosorbent assays (ELISAs), radioimmunoassays (RIAs), and fluorescence
activated cell
sorting (FACS).
Host cells transformed with NSEQ or polynucleotide sequences encoding PSEQ may
be
cultured under conditions suitable for the expression and recovery of the
protein from cell culture.
The protein produced by a transformed cell may be secreted or retained
intracellularly depending
on the sequence and/or the vector used. As will be understood by those of
skill in the art,
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expression vectors containing polynucleotides of NSEQ or polynucleotides
encoding PSEQ may
be designed to contain signal sequences which direct secretion of PSEQ or
polypeptides encoded
by NSEQ through a prokaryotic or eukaryotic cell membrane.
In addition, a host cell strain may be chosen for its ability to modulate
expression of the
S inserted sequences or to process the expressed protein in the desired
fashion. Such modifications
of the polypeptide include, but are not limited to, acetylation,
carboxylation, glycosylation,
phosphorylation, lipidation, and acylation. Post-translational processing
which cleaves a "prepro"
form of the protein may also be used to specify protein targeting, folding,
and/or activity.
Different host cells which have specific cellular machinery and characteristic
mechanisms for
post-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and WI38), are
available from
the American Type Culture Collection (ATCC, Manassas VA) and may be chosen to
ensure the
correct modification and processing of the foreign protein.
In another embodiment of the invention, natural, modified, or recombinant NSEQ
or
nucleic acid sequences encoding PSEQ are ligated to a heterologous sequence
resulting in
translation of a fusion protein containing heterologous protein moieties in
any of the
aforementioned host systems. Such heterologous protein moieties facilitate
purification of fusion
proteins using commercially available affinity matrices. Such moieties
include, but are not limited
to, glutathione S-transferase (GST), maltose binding protein (MBP),
thioredoxin (Trx), calmodulin
binding peptide (CBP), 6-His, FLAG, c-myc, hemagglutinin (HA) and monoclonal
antibody
epitopes..
In another embodiment, NSEQ or sequences encoding PSEQ are synthesized, in
whole or
in part, using chemical methods well known in the art. (See, e.g., Caruthers,
M.H. et al. ( 1980)
Nucleic Acids Symp. Ser. (7):215-223; Horn, T. et al. (1980) Nucleic Acids
Symp. Ser.
(7):225-232; and Ausubel, supra). Alternatively, PSEQ or a polypeptide
sequence encoded by
2S NSEQ itself, or a fragment thereof, may be synthesized using chemical
methods. For example,
peptide synthesis can be performed using various solid-phase techniques
(Roberge, J.Y. et al.
(1995) Science 269:202-204). Automated synthesis may be achieved using the
AB1431A Peptide
synthesizer (PE Biosystems, Foster City CA). Additionally, PSEQ or the amino
acid sequence
encoded by NSEQ, or any part thereof, may be altered during direct synthesis
and/or combined
with sequences from other proteins, or any part thereof, to produce a
polypeptide variant.
In another embodiment, the invention provides a substantially purified
polypeptide
comprising the amino acid sequence selected from the group consisting of SEQ
ID NO: 6, or
fragments thereof.
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DIAGNOSTICS and THERAPEUTICS
The genes of the invention can be used in diagnosis, prevention, treatment,
and evaluation
of therapies for diseases related to neurotransmitter processing, particularly
neurological and
hormone-related diseases, more particularly Parkinson's disease,
schizophrenia, epilepsy, female
reproductive disorders, and attention deficit disorder. Further, the amino
acid sequences encoded
by the new genes are potential therapeutic proteins and targets of
therapeutics against these
diseases.
In one preferred embodiment, the NSEQ or the polynucleotides encoding PSEQ are
used
for diagnostic purposes to determine the absence, presence, and excess
expression of PSEQ, and
to monitor regulation of the levels of mRNA or the polypeptides encoded by
NSEQ during
therapeutic intervention. The polynucleotides may be at least 18 nucleotides
long, complementary
RNA and DNA molecules, branched nucleic acids, and peptide nucleic acids
(PNAs).
Alternatively, the polynucleotides are used to detect and quantitate gene
expression in samples in
which expression of PSEQ or the polypeptides encoded by NSEQ are correlated
with disease.
Additionally, NSEQ or the polynucleotides encoding PSEQ can be used to detect
genetic
polymorphisms associated with a disease. These polymorphisms may be detected
at the transcript
cDNA or genomic level.
The specificity of the probe, whether it is made from a highly specific
region, e.g., the 5'
regulatory region, or from a less specific region, e.g., a conserved motif,
and the stringency of the
hybridization or amplification (maximal, high, intermediate, or low), will
determine whether the
probe identifies only naturally occurring sequences encoding PSEQ, allelic
variants, or related
sequences.
Probes may also be used for the detection of related sequences, and should
preferably
have at least 50% sequence identity to any of the NSEQ or PSEQ-encoding
sequences.
Means for producing specific hybridization probes for DNAs encoding PSEQ
include the
cloning of NSEQ or polynucleotide sequences encoding PSEQ into vectors for the
production of
mRNA probes. Such vectors are known in the art, are commercially available,
and may be used to
synthesize RNA probes in vitro by means of the addition of the appropriate RNA
polymerases and
the appropriate labeled nucleotides. Hybridization probes may be labeled by a
variety of reporter
groups, for example, by radionuclides such as'ZP or'sS, or by enzymatic
labels, such as alkaline
phosphatase coupled to the probe via avidin/biotin coupling systems, by
fluorescent labels and the
like. The polynucleotide sequences encoding PSEQ may be used in Southern or
northern analysis,
dot blot, or other membrane-based technologies; in PCR technologies;and in
microarrays utilizing
fluids or tissues from patients to detect altered PSEQ expression. Such
qualitative or quantitative
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methods are well known in the art.
NSEQ or the nucleotide sequences encoding PSEQ can be labeled by standard
methods
and added to a fluid or tissue sample from a patient under conditions suitable
for the formation of
hybridization complexes. After a suitable incubation period, the sample is
washed and the signal
is quantitated and compared with a standard value. If the amount of signal in
the patient sample is
significantly altered in comparison to the standard value then the presence of
altered levels of
nucleotide sequences of NSEQ and those encoding PSEQ in the sample indicates
the presence of
the associated disease. Such assays may also be used to evaluate the efficacy
of a particular
therapeutic treatment regimen in animal studies, in clinical trials, or to
monitor the treatment of an
individual patient.
Once the presence of a disease is established and a treatment protocol is
initiated,
hybridization or amplification assays can be repeated on a regular basis to
determine if the level of
expression in the patient begins to approximate that which is observed in the
normal subject. The
results obtained from successive assays may be used to show the efficacy of
treatment over a
period ranging from several days to months.
The polynucleotides may be used for the diagnosis of a variety of diseases
associated with
neurotransmitter processing such as neurological disorders including, but not
limited to, akathesia,
Alzheimer's disease, amnesia, amylotrophic lateral sclerosis, bipolar
disorder, catatonia, cerebral
neoplasms, dementia, depression, diabetic neuropathy, Down's syndrome, tardive
dyskinesia,
dystonias, epilepsy, Huntington's disease, peripheral neuropathy, multiple
sclerosis,
neurofibromatosis, Parkinson's disease, paranoid psychoses, postherapeutic
neuralgia,
schizophrenia, and Tourette's disorder; and reproductive disorders including,
but not limited to,
disorders of prolactin production, tubal disease, ovulatory defects,
endometriosis, disruptions of
the estrous cycle, disruptions of the menstrual cycle, polycystic ovary
syndrome, ovarian
hyperstimulation syndrome, endometrial and ovarian tumors, uterine fibroids,
autoimmune
disorders, ectopic pregnancies, and teratogenesis, cancer of the breast,
testis, and prostate,
fibrocystic breast disease, galactorrhea disruptions of spermatogenesis,
abnormal sperm
physiology, prostatitis, Peyronie's disease, and gynecomastia.
Alternatively, the polynucleotides may be used as targets in a microarray. The
microarray
can be used to monitor the expression level of large numbers of genes
simultaneously and to
identify splice variants, mutations, and polymorphisms. This information may
be used to
determine gene function, to understand the genetic basis of a disease, to
diagnose a disease, and to
develop and monitor the activities of therapeutic agents.
1n yet another alternative, polynucleotides may be used to generate
hybridization probes
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useful in mapping the naturally occurring genomic sequence. Fluorescent in
situ hybridization
(FISH) may be correlated with other physical chromosome mapping techniques and
genetic map
data. (See, e.g., Heinz-Ulrich, et al. ( 1995) in Meyers, supra, pp. 965-968).
In another embodiment, antibodies which specifically bind PSEQ may be used for
the
diagnosis of diseases characterized by the over- or under-expression of PSEQ
or polypeptides
encoded by NSEQ. Alternatively, one may use competitive drug screening assays
in which
neutralizing antibodies capable of binding PSEQ or the polypeptides encoded by
NSEQ
specifically compete with a test compound for binding the polypeptides. In
this manner,
antibodies can be used to detect the presence of any peptide which shares one
or more antigenic
determinants with PSEQ or the polypeptides encoded by NSEQ. Diagnostic assays
for PSEQ or
the polypeptides encoded by NSEQ include methods which utilize the antibody
and a label to
detect PSEQ or the polypeptided encoded by NSEQ in human body fluids or in
extracts of cells or
tissues. A variety of protocols for measuring PSEQ or the polypeptides encoded
by NSEQ,
including ELISAs, RIAs, and FRCS, are well known in the art and provide a
basis for diagnosing
altered or abnormal levels of the expression of PSEQ or the polypeptides
encoded by NSEQ.
Normal or standard values for PSEQ expression are established by combining
body fluids or cell
extracts taken from normal subjects, preferably human, with antibody to PSEQ
or a polypeptide
encoded by NSEQ under conditions suitable for complex formation The amount of
standard
complex formation may be quantitated by various methods, preferably by
photometric means.
Quantities of PSEQ or the polypeptides encoded by NSEQ expressed in subject,
control, and
disease samples from biopsied tissues are compared with the standard values.
Deviation between
standard and subject values establishes the parameters for diagnosing or
monitoring disease.
In another aspect, the polynucleotides and polypeptides of the present
invention can be
employed for treatment or the monitoring of therapeutic treatments for the
diseases specified
above. The polynucleotides of NSEQ or those encoding PSEQ, or any fragment or
complement
thereof, may be used for therapeutic purposes. In one aspect, the complement
of the
polynucleotides ofNSEQ or those encoding PSEQ may be used in situations in
which it would be
desirable to block the transcription or translation of the mRNA.
Expression vectors derived from retroviruses, adenoviruses, or herpes or
vaccinia viruses,
or from various bacterial plasmids, may be used for delivery of nucleotide
sequences to the
targeted organ, tissue, or cell population. Methods which are well known to
those skilled in the art
can be used to construct vectors to express nucleic acid sequences
complementary to the
polynucleotides encoding PSEQ. (See, e.g., Sambrook, supra; and Ausubel,
supra.)
Genes having polynucleotide sequences of NSEQ or those encoding PSEQ can be
turned
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off by transforming a cell or tissue with expression vectors which express
high levels of a
polynucleotide, or fragment thereof, encoding PSEQ. Such constructs may be
used to introduce
untranslatable sense or antisense sequences into a cell. Oligonucleotides
derived from the
transcription initiation site, e.g., between about positions -10 and +10 from
the start site, are
preferred. Similarly, inhibition can be achieved using triple helix base-
pairing methodology.
Triple helix pairing is useful because it causes inhibition of the ability of
the double helix to open
sufficiently for the binding of polymerises, transcription factors, or
regulatory molecules. Recent
therapeutic advances using triplex DNA have been described in the literature.
(See, e.g., Gee, J.E.
et al. ( 1994) in Huber, B.E. and B.1. Carr, Molecular and Immunologic
Aonroaches, Futura
Publishing Co., Mt. Kisco NY, pp. 163-177.) Ribozymes, enzymatic RNA
molecules, may also be
used to catalyze the specific cleavage of RNA.
RNA molecules may be modified to increase intracellular stability and half
life. Possible
modifications include, but are not limited to, the addition of flanking
sequences at the 5' and/or 3'
ends of the molecule, or the use of phosphorothioate or 2' O-methyl rather
than phosphodiesterase
linkages within the backbone of the molecule. This concept is inherent in the
production of PNAs
and can be extended in all of these molecules by the inclusion of
nontraditional bases such as
inosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-, and
similarly modified forms
of adenine, cytidine, guanine, thymine, and uridine which are not as easily
recognized by
endogenous endonucleases.
Many methods for introducing vectors into cells or tissues are available and
equally
suitable for use in vivo, in vitro, and ex vivo. For ex vivo therapy, vectors
may be introduced into
stem cells taken from the patient and clonally propagated for autologous
transplant back into that
same patient. Delivery by transfection, by liposome injections, or by
polycationic amino polymers
may be achieved using methods which are well known in the art. (See, e.g.,
Goldman, C.K. et al.
( 1997) Nature Biotechnology I 5:462-466.)
Further, an antagonist or antibody of a polypeptide of PSEQ or encoded by NSEQ
may be
administered to a subject to treat or prevent a disease related to synthesis
and release of dopamine
and norepinephrine with increased expression or activity of PSEQ. An antibody
which
specifically binds the polypeptide may be used directly as an antagonist or
indirectly as a targeting
or delivery mechanism for bringing a pharmaceutical agent to cells or tissue
which express the
polypeptide.
Antibodies to PSEQ or a polypeptide encoded by NSEQ may also be generated
using
methods that are well known in the art. Such antibodies may include, but are
not limited to,
polyclonat, monoclonal, chimeric, and single chain antibodies, Fab fragments,
and fragments
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produced by a Fab expression library. Neutralizing antibodies (i.e., those
which inhibit dimer
formation) are especially preferred for therapeutic use. Monoclonal antibodies
to PSEQ may be
prepared using any technique which provides for the production of antibody
molecules by
continuous cell lines in culture. These include, but are not limited to, the
hybridoma technique, the
human B-cell hybridoma technique, and the EBV-hybridoma technique. In
addition, techniques
developed for the production of chimeric antibodies can be used. (See, e.g.,
Meyers, supra.)
Alternatively, techniques described for the production of single chain
antibodies may be
employed. Antibody fragments which contain specific binding sites for PSEQ or
the polypeptide
sequences encoded by NSEQ may also be generated.
Various immunoassays may be used for screening to identify antibodies having
the
desired specificity. Numerous protocols for competitive binding or
immunoradiometric assays
using either polyclonal or monoclonal antibodies with established
specificities are well known in
the art.
Yet further, an agonist of a polypeptide of PSEQ or that encoded by NSEQ may
be
administered to a subject to treat or prevent a cancer associated with
decreased expression or
activity of the polypeptide.
An additional aspect of the invention relates to the administration of a
pharmaceutical or
sterile composition, in conjunction with a pharmaceutically acceptable
carrier, for any ofthe
therapeutic effects discussed above. Such pharmaceutical compositions may
consist of
polypeptides of PSEQ or those encoded by NSEQ, antibodies to the polypeptides,
and mimetics,
agonists, antagonists, or inhibitors ofthe polypeptides. The compositions may
be administered
alone or in combination with at least one other agent, such as a stabilizing
compound, which may
be administered in any sterile, biocompatible pharmaceutical carrier
including, but not limited to,
saline, buffered saline, dextrose, and water. The compositions may be
administered to a patient
alone, or in combination with other agents, drugs, or hormones.
The pharmaceutical compositions utilized in this invention may be administered
by any
number of routes including, but not limited to, oral, intravenous,
intramuscular, intra-arterial,
intramedullary, intrathecal, intraventricular, transdermal, subcutaneous,
intraperitoneal, intranasal,
enteral, topical, sublingual, or rectal means.
In addition to the active ingredients, these pharmaceutical compositions may
contain
suitable pharmaceutically-acceptable carriers comprising excipients and
auxiliaries which
facilitate processing of the active compounds into preparations which can be
used
pharmaceutically. Further details on techniques for formulation and
administration may be found
in the latest edition of ReminQton's Pharmaceutical Sciences (Maack Publishing
Co. Easton PA).
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For any compound, the therapeutically effective dose can be estimated
initially either in
cell culture assays, e.g., of neoplastic cells or in animal models such as
mice, rats, rabbits, dogs, or
pigs. An animal model may also be used to determine the appropriate
concentration range and
route of administration. Such information can then be used to determine useful
doses and routes
for administration in humans.
A therapeutically effective dose refers to that amount of active ingredient,
for example,
polypeptides of PSEQ or those encoded by NSEQ, or fragments thereof,
antibodies of the
polypeptides, and agonists, antagonists or inhibitors of the polypeptides,
which ameliorates the
symptoms or condition. Therapeutic efficacy and toxicity may be determined by
standard
pharmaceutical procedures in cell cultures or with experimental animals, such
as by calculating the
EDS° (the dose therapeutically effective in 50% of the population) or
LDs° (the dose lethal to SO%
of the population) statistics.
Any of the therapeutic methods described above may be applied to any subject
in need of
such therapy, including, for example, mammals such as dogs, cats, cows,
horses, rabbits,
I S monkeys, and most preferably, humans.
EXAMPLES
1t is understood that this invention is not limited to the particular
methodology, protocols,
and reagents described, as these may vary. It is also understood that the
terminology used herein
is for the purpose of describing particular embodiments only, and is not
intended to limit the scope
of the present invention which will be limited only by the appended claims.
The examples below
are provide to illustrate the subject invention and are not included for the
purpose of limiting the
invention.
I. PGANNOTOI cDNA Library Construction
For purposes of example, the preparation of the PGANNOTOI library is
described. The
PGANNOTO1 cDNA library was constructed using polyA RNA isolated from
paraganglionic
tumor tissue removed from the intra-abdominal region of a 46-year-old
Caucasian male during
exploratory laparotomy. Pathology indicated a benign paraganglioma and was
associated with a
grade 2 renal cell carcinoma, clear cell type, which did not penetrate the
capsule. Family history
included cerebrovascular disease, atherosclerotic coronary artery disease, a
myocardial infarction,
and type II diabetes.
The frozen tissue was homogenized and lysed in guanidinium isothiocyanate
solution
using a Poiytron homogenizes (PT-3000; Brinkmann Instruments, Westbury NJ).
The lysate was
centrifuged over a 5.7 M CsCI cushion using an SW28 rotor in a BL8-70M
ultracentrifuge
(Beckman Coulter, Fullerton CA) for 18 hours at 25,000 rpm at ambient
temperature. The RNA
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was extracted twice with acid phenol, pH 4.0, following an RNA isolation
protocol (Stratagene, La
Jolla CA) , precipitated using 0.3 M sodium acetate and 2.5 volumes of
ethanol, resuspended in
DEPC-treated water and treated with DNase for I 5 min at 37°C. The
reaction was stopped with
an equal volume of acid phenol and the RNA was isolated using the OLIGOTEX kit
(Qiagen ,
Chatsworth CA) and used to construct the cDNA library.
The RNA was handled according to the recommended protocols in the SUPERSCRIPT
Plasmid system (Life Technologies, Gaithersburg MD), and cDNAs were ligated
into pSport I
plasmid (Life Technologies). The plasmid was subsequently transformed into
DHSa competent
cells (Life Technologies).
II Isolation and Sequencing of cDNA Clones
Plasmid DNA was released from the cells and purified using the M1NIPREP kit
(Edge
Biosystems, Gaithersburg MD). This kit consists of a 96 well block with
reagents for 960
purifications. The recommended protocol was employed except for the following
changes: 1) the
96 wells were each filled with only 1 m) of sterile Terrific Broth (Life
Technologies) with
carbenicillin at 25 mg/L and glycerol at 0.4%; 2) the bacteria were cultured
for 24 hours after the
wells were inoculated and then lysed with 60 ~l of lysis buffer; 3) a
centrifugation step employing
the GS-6R (Beckman Coulter) at 2900 rpm for 5 min was performed before the
contents of the
block were added to the primary filter plate; and 4) the optional step of
adding isopropanol to
TRIS buffer was not routinely performed. After the last step in the protocol,
samples were
transferred to a 96-well block for storage.
Alternative methods of purifying plasmid DNA include the use of MAGIC
MINIPREPS
system (Promega, Madison WI) or QIAWELL-$ Plasmid, QIAWELL PLUS DNA and
QIAWELL ULTRA DNA purification systems (Qiagen).
The cDNAs were prepared using a MICROLAB 2200 system (Hamilton, Reno NV) in
combination with four DNA ENGINE thermal cyclers (PTC200; MJ Research,
Watertown MA)
and sequenced by the method of Sanger F and AR Coulson ( 1975; J Mol Biol
94:441 f) using ABI
PRISM 377 or 373 DNA sequencing systems (PE Biosystems).
III. Selection, Assembly, and Characterization of Seguences
The sequences used for coexpression analysis were assembled from EST
sequences, 5'
and 3' longread sequences, and full length coding sequences. Selected
sequences were expressed
in at least three cDNA libraries.
The assembly process is described as follows. EST sequence chromatograms were
processed and verified. Quality scores were obtained using PHRED (Swing, B. et
al. (1998)
Genome Res. 8:175-185; Swing, B. and P. Green (1998) Genome Res. 8:186-194).
The edited
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sequences were loaded into a relational database management system (RDBMS).
The EST
sequences were clustered into an initial set of bins using BLAST with a
product score of S0. All
clusters of two or more sequences were created as bins. The overlapping
sequences represented in
a bin correspond to the sequence of a transcribed gene.
Assembly of the component sequences within each bin was performed using a
modification of Phrap, a publicly available program for assembling DNA
fragments (Green, P.
University of Washington, Seattle WA). Bins that showed 82% identity from a
local pair-wise
alignment between any of the consensus sequences were merged.
Bins were annotated by screening the consensus sequence in each bin against
public
databases, such as GBPRI and GENPEPT from NCBI. The annotation process
involved a FASTn
screen against the GBPRI database in GENBANK. Those hits with a percent
identity of greater
than or equal to 70% and an alignment length of greater than or equal to 100
base pairs were
recorded as homolog hits. The residual unannotated sequences were screened by
FASTx against
GENPEPT. Those hits with an E value of less than or equal to 10-e are recorded
as homolog hits.
Sequences were then reclustered using BLASTn and Cross-Match, a program for
rapid
protein and nucleic acid sequence comparison and database search (Green, P.
University of
Washington, Seattle WA), sequentially. Any BLAST alignment between a sequence
and a
consensus sequence with a score greater than I50 was realigned using cross-
match. The sequence
was added to the bin whose consensus sequence gave the highest Smith-Waterman
score amongst
local alignments with at least 82% identity. Non-matching sequences created
new bins. The
assembly and consensus generation processes were performed for the new bins.
IV. Known Genes Used for Identifying New Genes Associated with
Neurotransmitter
Processing
Any of ten neurotransmitter-processing-specific genes were used to identify
new Incyte
genes, using the coexpression analysis method described above. The genes
included L-tyrosine
hydroxylase (TH), aromatic amino acid decarboxylase (AADC), dopamine ~i
hydroxylase (DBH),
nicotinic acetylcholine receptor a3 subunit precursor (nAchR-a3),
secretogranin I and II, Rab3a,
human cocaine and amphetamine regulated transcript (hCART), vesicular
monoamine transporter 1
(hVMATI), ARIX homeodomain protein, and the like. Other known genes that have
potential
association with dopamine and norepinephrine pathways include endothelin
converting enzymes
(ECEs), rapt-interacting protein 8 (RPIPB), neuron-specific growth-associated
protein (SCG10),
and "Delta-like" putative homeotic protein (dlk).
L-tyrosine hydroxylase (TH} catalyzes the conversion of L-tyrosine to L-dopa,
which is the
first and rate-limiting step in the synthesis of the catecholamines dopamine,
norepinephrine and
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epinephrine (Nagatsu, T. (1995) Essays Biochem. 30:15-35). TH activity is
regulated in several
ways including transcriptionally, by alternative mRNA splicing, and by
regulation of mRNA
stability. Aromatic amino acid decarboxylase (AADC) catalyzes the
decarboxylation of L-tyorsine,
L-tryptophan, L-phenylalanine and L-dopa. Decarboxylation of L-dopa yields
dopamine (Webster
and Jordan, supra), and increased levels of AADC enhanced dopamine synthesis
in vivo (Kaddis et
al., su ra). Dopamine ~i hydroxylase (DBH) converts dopamine to norepinephrine
(Wu, H.J. et al.
(1990) J. Neurochem. 55:97-105). Nicotinic acetylcholine receptor a3 subunit
precursor (nAchR-
a3) is a subunit in the ligand-gated sodium ion channel triggered by nicotine
and acetylcholine.
Prolonged stimulation of nAchR with nicotine or acetylcholine induces TH and
DBH (Patrick, R.L.
and J.D. Barchas (i976) J. Pharmacol. Exp: Ther. 197:181-196). Secretogranins
are vesicle
associated proteins. Secretogranin I is found in norepinephrine-containing
vesicles (Bressler, J.P.
et al. ( 1996) J. Neurosci. Res. 46:678-685). Secretogranin I1 is a precursor
for secretoneurin, which
stimulates dopamine release (Agneter, E. et al. (1995) J. Neurochem. 65:622-
625; Fischer-Colbrie,
R. et al. ( 1995) Prog. Neurobiol. 46:49-70). Secretogranin II occurs in
dopamine- and
norepinephrine-containing vesicles in neurons (Goodall, A.R. et al. ( 1997) J.
Neurochem. 68:1542-
1552). Rab3a is a member of the Ras GTPase family. Rab3a is associated with
large dense core
vesicles and partially co-localizes with DBH (Johannes, L. et al. (1994) Embo.
J. 13:2029-2037;
Darchen, F. et al. (1995) J. Cell Sci. 108: 1639-1649), and is one of the
factors controlling Ca2+-
dependent exocytosis (Johannes et al., supra). Human cocaine and amphetamine
regulated
transcript (hCART) is a signal peptide protein and is specifically induced in
neurons exposed to
cocaine and amphetamine (Douglass, J. and S. Daoud (1996) Gene 169:241-245).
Cocaine blocks
re-uptake ofdopamine from the synapse (Reith, M.E, et al. (1997) Eur. J.
Pharmacol. 324:1-10 as
does amphetamine, which also induces dopamine release (Jones, S.R. et al.
(1998) J. Neurosci.
I 8:1979-1986). Vesicular monoamine transporter 1 (hVMATI ) packages the
neurotransmitter
monoamines into large dense core vesicles for exocytosis at the synapse (Liu,
Y. et al. (1994) J.
Cell Biol. 127:1419-1433). ARIX is a human homeodomain protein which is
specifically
expressed in noradrenergic, DBH-positive tissues and in cell lines derived
from those tissues
(Zeilmer, E. et al. ( 1995) J. Neurosci. 15:8109-8120). ARIX regulates the
transcriptional activities
of the DBH promoter and the promoter of the tyrosine hydroxylase gene, two of
the promoters
which control the specific expression of catecholamine biosynthetic genes.
The endothelins, ET-1, ET-2, and ET-3, are a class of secreted peptides that
are processed
from inactive precursor peptides. The third and final processing step leading
to the active ET is
catalyzed by a member of the endothelin converting enzymes (ECEs). The ET's
were known
vasoactive agents, potential neurotransmitters, and potential growth factors
(Barnes, K. and A.J.
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Turner (1997) Neurochem. Res. 22:1033-1040). ET-I was shown to enhance
norepinephrine
pressor response (Kita, S. et al. (1998) J. Cardiovasc. Pharmacol. 31:5119-
S121), and ET-1 and
ET-3 to induce dopamine release (Horie, K. et al. (1995) Life Sci. 57:735-741;
Shibaguchi, H, et
al. (1997) Cell Mol. Neurobiol. 17:471-481). ET-2, however, has not been shown
in published
literature to be associated with norepinephrine or dopamine processing. Rap2-
interacting protein 8
(RPIPB) was discovered using a yeast two-hybrid screen of mouse brain cDNAs
using the small
GTP-binding protein Rap2 as bait (Janoueix-Lerosey, I. et al. ( 1998) Eur. J.
Biochem. 252(2):290-
298). Rap2 is a member of the Ras family, another member of which (Rab3a) was
known to be
involved in exocytosis of neurotransmitters. SCG 10 is a neuron-specific
growth-associated protein
which belongs to the stathmin family. SCG10 functions in increasing the
breakdown of
microtubules in the neural growth cone (Riederer, B. M. et al. (1997) Proc.
Natl. Acad. Sci.
91:741-745). dlk is a "Delta-like" putative homeotic protein thought to be
involved in
neuroendocrine differentiation (Laborda, J. et al. ( 1993) J. Biol. Chem.
268:3817-3820). It
belongs to the epidermal growth factor receptor family (Lee, Y.L. et al.
(1995) Biochim. Biophys.
Acta 1261:223-232) and is induced by growth hormone (Carlsson, C. et al.
(1997) Endocrinology
138:3940-3948) and regulated through ErbB3 (Edman, C.F. et al. (1997) Biochem.
J. 323:113-
118).
Table 4 shows the five genes that have the strongest association with TH, a
gene involved
in the synthesis and release of dopamine, and norepinephrine. These
coexpressed genes are
presented with their p-values. The column headings have the following
meanings:
P-value The probability that the observed number of co-occurrences is
due to chance using the Fisher Exact method.
Associated gene A gene that shows significant co-expression with the target.
Occurs The number of libraries in which the associated gene occurs.
Both occur The number of libraries in which both the target gene and the co-
expressed gene occur.
Target only The number of libraries in which only the target gene occurs.
Associated only The number of libraries in which only the associated gene
occurs.
Neither The number of libraries in which neither the target gene nor the
associated gene occur.
Table 4 Co-expression results for TH
P-value Associated gene Occurs Both Target Associated Neither
occur only only
1.47E-13 DBH 12 9 6 3 503
6.IlE-I1 nAchR-a3 13 8 7 5 501
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6.22E-10 Secretogranin I 45 I 1 4 34 472
3.51 E-09 Human dlk mRNA 52 11 4 41 465
1.01 E-08 Homolog to ECE I 3 7 8 6 500
As a target, TH occurred in 15 of 522 cDNA libraries studied, and showed
strong
coexpression with genes known to be related to neurotransmitter processing and
other known
genes, such as DBH, nAchR, secretogranin I, human dlk mRNA for putative
homeotic protein, and
a homolog of endothelin converting enzyme (ECE). These results are shown in
Table 4 with
association probability in the range of 1.58E-8 to 1.53E-13.
Similar results were observed when the other nine known genes, AADC, DBH,
nAchR-a3,
secretogranin I and II, Rab3a, hCART, hVMATI, and ARIX were taken as target
genes. These
results indicate that the coexpression method was successful in identifying
association of the known
genes among themselves and with the known genes specified above, and thus was
effective in
identifying genes that are associated with the synthesis and release of
neurotransmitters such as
dopamine and norepinephrine.
V. Identification of New Genes Associated with Neurotransmitter Processing
We have identified five new genes by testing for the co-expression of their
cDNA with that
from genes known to be dopamine- and norepinephrine-related in the libraries
comprising
LIFESEQ database (Incyte Pharamaceuticals). The five genes show statistically
significant
associations with the known dopamine- and norepinephrine-related genes as
measured by a Fisher
exact test. These five new genes are potential therapeutic proteins and
therapeutic targets for the
treatment of diseases in which the levels of norepinephrine and/or dopamine
are perturbed from
their normal levels.
The new genes were identified from a total of41,419 assembled Incyte gene
sequences.
The degree of association was measured by probability values and has a cutoff
of p value less than
0.00001. This was followed by annotation and literature searches to insure
that the genes that
passed the probability test have strong association with known
neurotransmitter-processing-specifc
genes. This process was reiterated so that the initial 41419 genes were
reduced to the final five
genes associated with neurotransmitter processing. Details of identification
for the five genes are
presented in Tables 5 to 10. These tables show the five genes that were most
closely associated for
each target new gene as measured by coexpression using the Fisher Exact test.
The column
headings have the same meanings as in Example IV.
Table 5 Coexpression results for Incyte gene 621850
P-value Associated gene Occurs Both Target Associated Neither
occur only only
6.91E-09 DBH 12 6 4 6 SOS
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8.41 E-08 Somatostatin I 29 7 3 22 489
I .37E-07 SCG 10 49 8 2 41 470
2.25E-07 Secretogranin II 33 7 3 26 485
Incyte gene 621850 occurred in 10 of 522 cDNA libraries studied and showed
strong co-
expression with several of the known genes related to neurotransmitter
processing, including DBH,
somatostatin I and II, as shown in Table S. 621850 also showed strong
association with a SCG10.
SCG10 was shown in Example IV to be strongly associated with the known genes
related to
synthesis and release of dopamine. These results are consistent with the
notion that 621850 is
associated with neurotransmitter processing. .
Table 6 Coexpression
results for
Incyte gene
625839
P-value Associated Occurs Both TargetAssociatedNeither
gene
occuronly only
0 DBH 12 7 7 5 502
0 nAchR 13 7 7 6 501
0 TH type 4 15 7 7 8 499
0 Secretogranin 33 8 6 25 482
II
0 PARP 34 8 6 26 481
Incyte gene 625839 occurred in 14 of 522 cDNA libraries studied and showed
strong co-
expression with several of the known genes related to synthesis and release of
dopamine, and
norepinephrine, including DBH, nAchR, TH type 4, and secretogranin II, as
shown in Table 6.
625839 also showed strong association with PARP, a human poly(ADB-ribose)
polymerase which
is expressed during development in Drosophila (Uchida, K. et al. (1993)
90:3481-3485). These
results are consistent with the notion that 625839 is associated with
neurotransmitter processing.
Table 7 Coexpression results for Incyte gene 2405140
P-value Associated geneOccursBoth Target AssociatedNeither
occur only only
9.39E-06 DBH 12 3 0 9 509
1.22E-OS nAchR 13 3 0 10 508
1.94E-OSTH type 4 15 3 0 12 506
3.48E-OS AADC 18 3 0 15 503
3.48E-OS Homolog to predicted18 3 0 1 S 503
c. elegans protein
F25H2.8
Incyte gene 2405140 occurred in 3 of 522 cDNA libraries studied and showed
strong co-
expression with several of the known genes related to neurotransmitter
processing, including DBH,
nAchR, TH type 4, and AADC, as shown in Table 7. 2405140 also showed strong
association with
a homolog to a predicted C. elegans protein. F25H2.8. These results are
consistent with the notion
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that 2405140 is associated with neurotransmitter processing.
Table 8 Coexpression results for Incyte gene 2823339
P-value Associated gene Occurs Both Target Associated Neither
occur only only
2.20E-08 Secretogranin 1 45 9 3 36 473
4.51 E-08 Secretogranin ll 33 8 4 25 484
1.37E-07 hVMATI 8 5 7 3 506
1.56E-07 TH type 4 15 6 6 9 500
lncyte gene 2823339 occurred in 12 of 522 cDNA libraries studied and showed
strong co-
expression with several of the known genes related to neurotransmitter
processing, including
secretogranin I and ll, hVMATI and TH type 4, as shown in Tabie 8. These
results are consistent
with the notion that 2823339 is associated with neurotransmitter processing.
Table 9 Coexpression results for Incyte gene 2825861
P-value Associated gene Occurs Both Target Associated Neither
occur only only
0 DBH 12 3 0 9 509
0 TH type 4 15 3 0 12 506
0 Human factor X 29 3 0 26 492
Incyte gene 2825861 occurred in 3 of 522 cDNA libraries studied and showed
strong co-
expression with two of the known genes related to neurotransmitter processing,
including DBH and
TH type 4, as shown in Table 9. 2825861 also shows association with a blood
coagulation factor,
human factor X. These results are consistent with the notion that 2825861 is
associated with
neurotransmitter processing..
VI. New Genes Associated with Neurotransmitter Processing
Nucleic acids comprising nucleic acid sequences of SEQ ID NOs: I-5 of the
present
invention were identified from lncyte Clones 621850, 625839, 2405140, 2823339,
and 2825861,
respectively, and assembled as described in Example III. The nucleic acid
sequences were
translated and reading frames determined when possible. A full length protein
sequence, SEQ ID
NO: 6, was derived from SEQ ID NO: 4 after determining its coding frame. SEQ
ID NO: I-6 were
then annotated according to Example VII using BLAST and other motif search
tools against
databases of known molecules.
SEQ ID NO: 1 showed 99% sequence identity with a human brain-specific mltNA,
KIAA0604 (g3043731 ). In addition, a polypeptide sequence translated from SEQ
ID NO:1 showed
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about 77.3% sequence homology with the gene that encodes endothelin-converting
enzyme 2 of
Bos taurus, ECE-2. As described in Example IV, ECEs are known to be involved
in the pathway of
neurotransmitter processing.
SEQ ID NO: 2 showed 60% sequence identity from about nucleotide 80 to about
nucleotide 1818 with a human brain-specific gene, KIAA0604 (g3043731 ), and 61
% sequence
identity from about necleotide 144 to about nucleotide 1760 with a human mRNA
for endothelin-
converting enzyme 1, ECE-1 (gl 197803). ECE-1 is a metalloprotease known to be
associated with
the neurotransmitter processing.
SEQ ID N0:4 showed about 58% sequence identity from nucleotide 332 to
nucleotide
1393 with the gene which encodes the human heavy neurofilament subunit, NF-H
(g35028), a
homolog to intermediate filament (IF) proteins. The corresponding amino acid
sequence, SEQ ID
NO: 6, is 210 amino acids in length and has one potential N-glycosylation site
at N16; three
potential casein kinase II phosphorylation sites at S21, S 100, and S 114; one
potential
glycosaminoglycan attachment site at 5110; and two potential protein kinase C
phosphorylation
sites at S27 and S110.
VII. Homology Searching for Genes Associated with Neurotransmitter Processing
Polynucleotide sequences, SEQ ID NOs: 1-5, and polypeptide sequences, SEQ ID
NO: 6,
were queried against databases derived from sources such as GenBank and
SwissProt. These
databases, which contain previously identified and annotated sequences, were
searched for regions
of similarity using Basic Local Alignment Search Tool (BLAST; Altschul, S.F.
et al. (1990) J. Mol.
Biol. 215:403-410) and Smith-Waterman alignment (Smith, T. et al. ( 1992)
Protein Engineering
5:35-51 ). BLAST searched for matches and reported only those that satisfied
the probability
thresholds of 10''-5 or less for nucleotide sequences and 10'8 or less for
polypeptide sequences.
The polypeptide sequences were also analyzed for known motif patterns using
MOTIFS,
SPSCAN, BLIMPS, and Hidden Markov Mode) (HMM)-based protocols. MOTIFS
(Genetics
Computer Group, Madison WI) searches polypeptide sequences for patterns that
match those
defined in the Prosite Dictionary of Protein Sites and Patterns (Bairoch, A.
et al. ( 1997) Nucleic
Acids Res. 25:217-221 ), and displays the patterns found and their
corresponding literature
abstracts. SPSCAN (Genetics Computer Group, Madison, WI) searches for
potential signal
peptide sequences using a weighted matrix method (Nielsen, H. et al. (1997)
Prot. Eng. 10:1-6).
Hits with a score of 5 or greater were considered. BLIMPS uses a weighted
matrix analysis
algorithm to search for sequence similarity between the polypeptide sequences
and those
contained in BLOCKS, a database consisting of short amino acid segments, or
blocks, of 3-60
amino acids in length, compiled from the PROSITE database (Henikoff, S. and G.
J. Henikoff
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(1991) Nucleic Acids Res. 19:6565-6572; Bairoch et al., supra), and those in
PRINTS, a protein
fingerprint database based on non-redundant sequences obtained from sources
such as SwissProt,
GenBank, PIR, and NRL-3D (Attwood, T. K. et al. (1997) J. Chem. Inf. Comput.
Sci.
37:417-424). For the purposes of the present invention, the BLIMPS searches
reported matches
with a cutoff score of 1000 or greater and a cutoff probability value of 1.0 x
10'3. HMM-based
protocols were based on a probabilistic approach and searched for consensus
primary structures of
gene families in the protein sequences (Eddy, S.R. (1996) Cur. Opin. Str.
Biol. 6:361-365;
Sonnhammer, E.L.L. et al. (1997) Proteins 28:405-420). More than 500 known
protein families
with cutoff scores ranging from 10 to 50 bits were selected for use in this
invention.
VIII. Labeling and Use of Individual Hybridization Probes
Oligonucleotides are designed using state-of the-art software such as OLIGO
4.06
(National Biosciences) and labeled by combining 50 pmol of each oligomer, 250
,uCi of [y-'zp)
adenosine triphosphate (Amersham Pharmacia Biotech, Piscataway NJ), and T4
polynucleotide
kinase (NEN Life Science Products, Boston MA). The labeled oligonucleotides
are substantially
purified using a SEPHADEX G-25 superfine resin column (Amersham Pharmacia
Biotech). An
aliquot containing 10'counts per minute of the labeled probe is used in a
typical membrane-based
hybridization analysis of human genomic DNA digested with one of the following
endonucleases:
Ase I, Bgl II, Eco RI, Pst I, Xba 1, or Pvu II (NEN Life Science Products).
The DNA from each digest is fractionated on a 0.7 percent agarose gel and
transferred to
nylon membranes (NYTRAN PLUS, Schleicher & Schuell, Durham NH). Hybridization
is carried
out for 16 hours at 40°C. To remove nonspecific signals, blots are
sequentially washed at room
temperature under increasingly stringent conditions up to 0.1 x saline sodium
citrate and 0.5%
sodium dodecyl sulfate. After XOMAT AR film (Eastman Kodak, Rochester NY) is
exposed to
the blots for several hours, hybridization patterns are compared.
IX. Production of Specific Antibodies
SEQ ID NO: 6 substantially purified using polyacrylamide gel electrophoresis
(PAGE;
see, e.g., Harrington, M.G. (1990) Methods Enzymol. 182:488-495), or other
purification
techniques, is used to immunize rabbits and to produce antibodies using
standard protocols.
Alternatively, the amino acid sequence is analyzed using LASERGENE software
(DNASTAR Madison W1) to determine regions of high immunogenicity, and a
corresponding
oligopeptide is synthesized and used to raise antibodies by means known to
those of skill in the
art. Methods for selection of appropriate epitopes, such as those near the C-
terminus or in
hydrophilic regions are well described in the art. Typically, oligopeptides 15
residues in length are
synthesized using an ABI 431A Peptide synthesizer (PE Biosystems) using Fmoc-
chemistry and
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coupled to KLH (Sigma-Aldrich, St. Louis MO) by reaction with N-
maleimidobenzoyl-N-
hydroxysuccinimide ester to increase immunogenicity. Rabbits are immunized
with the
oligopeptide-KLH complex in complete Freund's adjuvant. Resulting antisera are
tested for
antipeptide activity by, for example, binding the peptide to plastic, blocking
with 1 % BSA,
reacting with rabbit antisera, washing, and reacting with radio-iodinated goat
anti-rabbit IgG.
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SEQUENCE LISTING
<110> INCYTE PHARMACEUTICALS, INC.
WALKER, Michael G.
VOLKMUTH, Wayne
KLINGLER, Tod M.
<120> GENES ASSOCIATED WITH NEUROTRANSMITTER PROCESSING
<130> PB-0003 PCT
<140> To Be Assigned
<141> Herewith
<150> 09/149,952
<151> 1998-09-O1
<160> 6
<170> PERL Program
<210> 1
<211> 3000
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte template ID No.: 621850CB1
<900> 1
gctgggagct ggcagcaacg tgggattcca gaaggggaca agacagctgt taggctcacg 60
cacgcagctg gagctggtct tagcaggtgc ctetctactg ctggctgcac tgcttctggg 120
ctgccttgtg gccctagggg tccagtacca cagagaccca tcccacagca cctgccttac 180
agaggcctgc attcgagtgg ctggaaaaat cctggagtcc ctggaccgag gggtgagccc 290
ctgtgaggac ttttaccagt tctcctgtgg gggctggatt cggaggaacc ccctgcccga 300
tgggcgttct cgctggaaca ccttcaacag cctctgggac caaaaccagg ccatactgaa 360
gcacctgctt gaaaacacca ccttcaactc cagcagtgaa gctgagcaga agacacagcg 920
cttctaccta tcttgcctac aggtggagcg cattgaggag ctgggagccc agccactgag 980
ggacctcatt gagaagattg gtggttggaa cattacgggg ccctgggacc aggacaactt 540
tatggaggtg ttgaaggcag tagcagggac ctacagggcc accccattct tcaccgtcta 600
catcagtgct gactctaaga gttccaacag caatgttatc caggtggacc agtctgggct 660
ctttctgccc tctcgggatt actacttaaa cagaactgcc aatgagaaag tgctcactgc 720
ctatctggat tacatggagg aactggggat gctgctgggt gggcggccca cctccacgag 780
ggagcagatg cagcaggtgc tggagttgga gatacagctg gccaacatca cagtgcccca 840
ggaccagcgg cgcgacgagg agaagatcta ccacaagatg agcatttcgg agctgcaggc 900
tctggcgccc tccatggact ggcttgagtc cctgtctttc ttgctgtcac cattggagtt 960
gagtgactct gagcctgtgg tggtgtatgg gatggattat ttgcagcagg tgtcagagct 1020
catcaaccgc acggaaccaa gcatcctgaa caattacctg atctggaacc tggtgcaaaa 1080
gacaacctca agcctggacc gacgctttga gtctgcacaa gagaagctgc tggagaccct 1140
ctatggcact aagaagtcct gtgtgccgag gtggcagacc tgcatctcca acacggatga 1200
cgcccttggc tttgctttgg ggtccctctt cgtgaaggcc acgtttgacc ggcaaagcaa 1260
agaaattgca gaggggatga tcagcgaaat ccggaccgca tttgaggagg ccctgggcaa 1320
gctggtttgg atggatgaga agacccgcca ggcagccaag gagaaagcag atgccatcta 1380
tgatatgatt ggtttcccag actttatcct ggagcccaaa gagctggatg atgtttatga 1440
cgggtacgaa atttctgaag attctttctt ccaaaacatg ttgaatttgt acaacttctc 1500
tgccaaggtt atggctgacc agctccgcaa gcctcccagc cgagaccagt ggagcatgac 1560
cccccagaca gtgaatgcct actaccttcc aactaagaat gagatcgtct tccccgctgg 1620
catcctgcag gcccccttct atgcccgcaa ccaccccaag gccctgaact tcggtggcat 1680
cggtgtggtc atgggccatg agttgacgca tgcctttgat gaccaagggc gcgagtatga 1790
caaagaaggg aacctgcggc cctggtggca gaatgagtcc ctggcagcct tccggaacca 1800
1/5
CA 02340617 2001-02-28
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cacggcctgc atggaggaac agtacaatca ataccaggtc aatggggaga ggctcaacgg 1860
ccgccagacg ctgggggaga acattgcgga caacgggggg ctgaaggctg cctacaatgc 1920
ttacaaagca tggctgagaa agcatgggga ggagcagcaa ctgccagccg tggggctcac 1980
caaccaccag ctcttcttcg tgggatttgc ccaggtgtgg tgctcggtcc gcacaccaga 2040
gagttctcac gaggggctgg tgaccgaccc ccacagccct gcccgcttcc gcgtgctggg 2100
cactctctcc aactcccgtg acttcctgcg gcacttcggc tgccctgtcg gctcccccat 2160
gaacccaggg cagctgtgtg aggtgtggta gacctggatc aggggagaaa tgcccagctg 2220
tcaccagacc tggggcagct ctcctgacaa agctgtttgc tcttgggttg ggaggaagca 2280
aatgcaagct gggctgggtc tagtccctcc cccccacagg tgacatgagt acagaccctc 2340
ctcaatcacc acattgtgcc tctgctttgg gggtgcccct gcctccagca gagcccccac 2400
cattcactgt gacatctttc cgtgtcaccc tgcctggaag aggtctgggt ggggaggcca 2460
gttcccatag gaaggagtct gcctcttctg tccccaggct cactcagcct ggcggccatg 2520
gggcctgccg tgcctgcccc actgtgaccc acaggcctgg gtggtgtacc tcctggactt 2580
ctccccaggc tcactcagtg cgcacttagg ggtggactca gctctgtctg gctcaccctc 2640
acgggctacc cccacctcac cctgtgctcc ttgtgccact gctcccagtg ctgctgctga 2700
ccttcactga cagctcctag tggaagccca agggcctctg aaagcctcct gctgcccact 2760
gtttccctgg gctgagaggg gaagtgcata tgtgtagcgg gtactggttc ctgtgtctta 2820
gggcacaagc ctttagcaaa tgattgattc tccctggaca aagcaggaaa gcagatagag 2880
cagggaaaag gaagaacaga gtttattttt acagaaaaga gggtgggagg gtgtggtctt 2990
ggcccttata ggaccctgtg ccaataaaca gacatgcatc cgtgaaaaaa aaaaaaaaaa 3000
<210> 2
<211> 2150
<212> DNA
<213> Homo Sapiens
<220>
<221> unsure
<222> 2107, 2116, 2124, 2133, 2137, 2190
<223> a or g or c or t, unknown, or other
<220>
<221> misc_feature
<223> Incyte template ID No.: 625839CT1
<400> 2
gcgagatcga gcgactgggc ccgcgaccca tgctagaggt catcgaggac tgcgggggct 60
gggacctggg cggcgcggag gagcgtccgg gggtcgcggc gcgatgggac ctcaaccggc 120
tgctgtacaa ggcgcagggc gtgtacagcg ccgccgcgct cttctcgctc acggtcagcc 180
tggacgacag gaactcctcg cgctacgtca tccgcattga ccaggatggg ctcaccctgc 240
cagagaggac cctgtacctc gctcaggatg aggacagtga gaagatcctg gcagcataca 300
gggtgttcat ggagcgagtg ctcagcctcc tgggtgcaga cgctgtggaa cagaaggccc 360
aagagatcct gcaagtggag cagcagctgg ccaacatcac tgtgtcagag tatgacgacc 420
tacggcgaga tgtcagctcc atgtacaaca aggtgacgct ggggcagctg cagaagatca 480
ccccccactt gcggtggaag tggctgctag accagatctt ccaggaggac ttctcagagg 540
aagaggaggt ggtgctgctg gcgacagact acatgcagca ggtgtcgcag ctcatccgct 600
ccacacccca ccgggtcctg cacaactacc tggtgtggcg cgtggtggtg gtcctgagtg 660
aacacctgtc cccgccattc cgtgaggcac tgcacgagct ggcacaggag atggagggca 720
gcgacaagcc acaggagctg gcccgggtct gcttgggcca ggcccaatcg ccactttggc 780
atggcgcttg gcgccctctt tgtacatgag cattctcagc tgccagcaaa gccaaggtgc 840
agcagctagt ggaagacatc aagtacatcc tgggccagcg cctggaggag ctggactgga 900
tggacgccga gaccagggct gctgctcggg gccaagctcc agtacatgat ggtgatggtc 960
ggctacccgg acttcctgct gaaacccgat gctgtggaca aggagtatga gtttgaggtc 1020
catgagaaga cctacttcaa gaacatcttg aacagcatcc gcttcagcat ccagctctgc 1080
agttaagaag attcggcagg aggtggacaa gtccacgtgg ctgctccccc cacaggcgct 1140
caatgcctac tatctaccca acaagaacca gatggtgttc cccgcgggca tcctgcagcc 1200
caccctgtac gaccctgact tcccacagtc tctcaactac gggggcatcg gcaccatcat 1260
tggacatgag ctgacccacg gctacgacga ctgggggggc cagtatgacc gctcagggaa 1320
cctgctgcac tggtggacgg aggcctccta cagccgcttc ctgcgaaagg ctgagtgcat 1380
cgtccgtctc tatgacaact tcactgtcta caaccagcgg gtgaacggga aacacacgct 1440
tggggagaac atcgcagata tgggcggcct caagctggcc taccacgcct atcagaagtg 1500
ggtgcgggag cacggcccag agcacccact tccccggctc aagtacacac atgaccagct 1560
2/5
CA 02340617 2001-02-28
WO 00/12685 PCT/US99/19615
cttcttcatt gcctttgccc agaactggtg catcaagcgg cggtcgcagt ccatctacct 1620
gcaggtgctg actgacaagc atgcccctga gcactacagg gtgctgggca gtgtgtccca 1680
gtttgaggag tttggccggg ctttccactg tcccaaggac tcacccatga accctgccca 1740
caagtgttcc gtgtggtgag cctggctgcc cgcctgcacg cccccactgc ccccgcacga 1800
atcacctcct gctggctacc ggggcaggca tgcacccggt gccagccccg ctctgggcac 1860
cacctgcctt ccagcccctc caggacccgg tccgcctgct gcccctcact tcaggagggg 1920
cctggagcag ggtgaggctg gactttgggg ggctgtgagg gaaatatact ggggtcccca 1980
gattctgctc taagggggcc agaccctctg ccaggctgga ttgtacgggc cccaccttcg 2040
ctgtgttctt gctgcaaagt ctggtcaata aatcactgca ctgttaaaaa aaaaaattag 2100
gggtgtntaa agtggntgga cacngggtca acntatnttn gaaccttttg 2150
<210> 3
<211> 512
<212> DNA
<213> Homo sapiens
<220>
<221> misc feature
<223> Incyte template ID No.: 2905140CB1
<400> 3
gagaaatgta taaaataatc gagaccggcg gaggcaggtc agagccgtct ggaatgcgcg 60
caccttcaac gaacaatgcc aacattgaag tcctcggttg gagtctgcac agttggagat 120
ctttggtgcc attttagaca tctttggatt tcatcaatca aactgactgc aattttccat 180
aaaaaccctg aatttgggtc agaaagtggg caaagtagat aaagatcatt cgagctgtct 290
tataagatga taaatagata tcctttcagg ccaacaatgc caaagtgcag ttttgtgatt 300
cccttccatg ggttctgaat gcagtgagtc gaaacgattt ctacatgttt tcccatggtt 360
taggaggtgt ctttacatac ttgtcaatag tagcctgacc tttttcccca tggagttgct 420
aagtgtgttt tgtttgttgc tttgagtact tttttcttgt tgtttgtgtg tgtgttgcac 480
aaaatacaca agaaaataaa aaaaaaaaaa as 512
<210> 4
<211> 1511
<212> DNA
<213> Homo sapiens
<220>
<221> misc_feature
<223> Incyte template ID No.: 2823339CB1
<400> 9
tccagagtgc tgaatttctg ggcagccagg gggctcttgc tctgctatgg gttgaagatt 60
cagcctgatc acttcttgtc aaagttgggg agtctgagct ggttctgggc ctagcaaggc 120
aatctactct gttaaagtcg atagagggag aagctgggga gaacactgca attttttcta 180
tgagccctct gtagagggtg ggtggtgggg gctccacaga ctgctccttg gttccacagg 240
ccatgcccgg aggtgctggc ttcgagccct tcagacaagt aaggtcagct ctgccccctc 300
ctgcttcact tgccctgtag ctgtggatgc tactactagg gaggtcctgc ctggggccct 360
gcaggtgtgc agctcagagc ccgctgagcc cccaggaacc cctcctgcct ctcacagcca 920
tctagatgca gctcctctgc ccactgttgt ctactctaaa ggacttcaga gaggctctcc 480
agcaggcgcc tgggactcgg accaaaatgg caactccaag cgtgctttgg gggaccctgc 540
cactcccacg gaaggtcctc gccgcccacc tccccgtcct ccctgtcggc tcagcatggg 600
ccgccgtcac aaactctgta gccctgaccc gggccaggcc aacaacagtg aaggcagcga 660
ccatgactac ctgcccttgg tgcggctgca ggaggcacca ggctccttcc gcctggacgc 720
gcccttctgc gccgctgtgc gcatctcgca ggagcgcctc tgccgtgcct cgccctttgc 780
cgtgcaccgc gccagcctca gccccacctc ggcctcattg ccctgggcac ttctgggccc 840
tggtgttggc cagggtgaca gtgccacggc ctcctgcagc ccgtccccca gctcgggctc 900
tgaggggcca ggccaggtgg acagtgggcg gggctcagac accgaggcct ccgatggggc 960
ggaagggctg ggcggcaccg acctgcgggg ccggacctgg gccactgccg tagcactcgc 1020
ctggctggag caccgatgcg ccgctgcctt cgacgagtgg gaactgacag cggccaaggc 1080
tgattgctgg ctgcgggccc agcacttgcc tgacggcctt gacctggccg ccctcaaggc 1140
cgcagcccga gggctcttcc tgctactgcg ccactgggac caaaacctgc agctacacct 1200
3/5
CA 02340617 2001-02-28
WO 00/I2685 PCT/US99/19615
gctgtgctac agcccagcga acgtgtgaag gctgccccct gctgcttggg ctggcgcccc 1260
acccaacaca ctcaagtcac tgccgcccag ggctggcctc ttggtgctgg gaaagtgtag 1320
gctggtgcca gcctgtcccc cactgcttct tactccctcc ctggagccct cttgccccca 1380
caaaaagtgc ctgcctgtgc tctctccctc tcctcccacc ccactcacac tcccctccat 1990
cctctgagct ccctgcaaca cagtggaagg gtagagagcc acagtcccca aatcctatgc 1500
aataaagtgc a 1511
<210> 5
<211> 697
<212> DNA
<213> Homo Sapiens
<220>
<221> misc_feature
<223> Incyte template ID No.: 2825861CT1
<900> 5
cggctcgagc ttccaaaaaa ctttcaatct tatgctgtgt caagatatca ctggctgttg 60
gtaaagtgct aatagcagaa tcagagagtg ctcatgtact tgctgattga aaatacatga 120
ttgacatgat tggaagtaca ctccagcttg ggtacagagt gagactgtct caaaaaaaca 180
acaacaaaaa agagacaaga ctatcttaca cttttttgtg gtttttccct tgcccttata 240
gcttccctac agtagtgggt aaccagtatc caactgaagg ctctgaaaag ggcacaaaga 300
cctcctagaa agagtacttc atctgatcaa cactaagaag ttcatctgtc tgctgctgct 360
gctgcggaca agctttatca ccagaaagtc atttataacg tttgcgtcaa taatgtctga 420
atggcttaaa gtaccatgca acattcattt gaatgttctg agtgatcaaa gtgaaatggc 980
tacaaccgga acacaatgaa gaggtacaga aaacaaaact caaacatccc aaggaaatgc 590
gtgttttgga acttttgggg agacagcatg gtgcagccac attccatgat tggctccagt 600
ggtgatccag gaagtgccta tgagtaagac ttgtatattt ctcatcactg ctctttcaac 660
ttctagcaca gcacctgaca tgtaataaat attcgca 697
<210> 6
<211> 210
<212> PRT
<213> Homo Sapiens
<220>
<221> misc feature
<223> Incyte template ID No.: 2823339CD1
<400> 6
Met Gly Arg Arg His Lys Leu Cys Ser Pro Asp Pro Gly Gln Ala
1 5 10 15
Asn Asn Ser Glu Gly Ser Asp His Asp Tyr Leu Pro Leu Val Arg
20 25 30
Leu Gln Glu Ala Pro Gly Ser Phe Arg Leu Asp Ala Pro Phe Cys
35 40 95
Ala Ala Val Arg Ile Ser Gln Glu Arg Leu Cys Arg Ala Ser Pro
50 55 60
Phe Ala Val His Arg Ala Ser Leu Ser Pro Thr Ser Ala Ser Leu
65 70 75
Pro Trp Ala Leu Leu Gly Pro Gly Val Gly Gln Gly Asp Ser Ala
80 85 90
Thr Ala Ser Cys Ser Pro Ser Pro Ser Ser Gly Ser Glu Gly Pro
95 100 105
Gly Gln Val Asp Ser Gly Arg Gly Ser Asp Thr Glu Ala Ser Asp
110 115 120
Gly Ala Glu Gly Leu Gly Gly Thr Asp Leu Arg Gly Arg Thr Trp
125 130 135
Ala Thr Ala Val Ala Leu Ala Trp Leu Glu His Arg Cys Ala Ala
190 145 150
4/5
CA 02340617 2001-02-28
WO 00/12685 PCT/US99/19615
Ala Phe Asp Glu Trp Glu Leu Thr Ala Ala Lys Ala Asp Cys Trp
155 160 165
Leu Arg Ala Gln His Leu Pro Asp Gly Leu Asp Leu Ala Ala Leu
170 175 180
Lys Ala Ala Ala Arg Gly Leu Phe Leu Leu Leu Arg His Trp Asp
185 190 195
Gln Asn Leu Gln Leu His Leu Leu Cys Tyr Ser Pro Ala Asn Val
200 205 210
5/5
