U.S. patent application number 11/170482 was filed with the patent office on 2006-05-04 for proteins related to schizophrenia and uses thereof.
Invention is credited to Paul E. Fraser, Peter H. St. George-Hyslop.
Application Number | 20060094037 11/170482 |
Document ID | / |
Family ID | 22863072 |
Filed Date | 2006-05-04 |
United States Patent
Application |
20060094037 |
Kind Code |
A1 |
St. George-Hyslop; Peter H. ;
et al. |
May 4, 2006 |
Proteins related to schizophrenia and uses thereof
Abstract
Presenilin Associated Membrane Protein (PAMP), and nucleic acids
encoding this protein, are provided. PAMP and PAMP nucleic acids
provide diagnostic and therapeutic tools for evaluating and
treating or preventing neurodevelopmental and neuropsychiatric
disorders. In a specific embodiment, mutations in PAMP are
diagnostic for schizophrenia. The invention further relates to
screening, particularly using high-throughput screens and
transgenic animal models, for compounds that modulate the activity
of PAMP and presenilins. Such compounds, or gene therapy with PAMP,
can be used in treating neurodevelopmental and neuropsychiatric
disorders, particularly schizophrenia. In addition, the invention
provides PAMP mutants, nucleic acids encoding for PAMP mutants, and
transgenic animals expressing PAMP mutants, which in a preferred
aspect result in biochemical, morphological, or neuropsychological
changes similar to those associated with schizophrenia.
Inventors: |
St. George-Hyslop; Peter H.;
(Toronto, CA) ; Fraser; Paul E.; (Toronto,
CA) |
Correspondence
Address: |
DARBY & DARBY P.C.
P. O. BOX 5257
NEW YORK
NY
10150-5257
US
|
Family ID: |
22863072 |
Appl. No.: |
11/170482 |
Filed: |
June 28, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09945258 |
Aug 31, 2001 |
6929919 |
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11170482 |
Jun 28, 2005 |
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60229889 |
Sep 1, 2000 |
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Current U.S.
Class: |
435/6.16 |
Current CPC
Class: |
G01N 2800/302 20130101;
G01N 33/6893 20130101; A61K 38/00 20130101; A01K 2217/05 20130101;
A61P 43/00 20180101; C07K 14/705 20130101; C12Q 1/6883 20130101;
G01N 2800/30 20130101; C12Q 2600/156 20130101; A61P 25/08 20180101;
G01N 2800/28 20130101; G01N 33/6896 20130101; G01N 2500/00
20130101 |
Class at
Publication: |
435/006 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Claims
1. A method for detecting a mutation in presenilin associated
membrane protein (PAMP) associated with a neuropsychiatric or
neurodevelopmental disorder, which method comprises detecting a
variation in a sequence of a gene encoding PAMP obtained from an
individual diagnosed with or suspected of having said disorder.
2. The method of claim 1, wherein the disorder is
schizophrenia.
3. A method for diagnosing individuals predisposed to or having a
neuropsychiatric or neurodevelopmental disorder, which method
comprises detecting a mutation in a gene encoding PAMP obtained
from an individual.
4. The method of claim 3, wherein the disorder is
schizophrenia.
5. The method according to claim 3, wherein detection of the
mutation comprises measuring a level of transcriptional activity of
the gene.
6. he method according to claim 3, wherein detection of the
mutation comprises measuring PAMP activity.
7. The method of claim 6, wherein said PAMP activity comprises PAMP
expression level or activity of a product of a PAMP modified
substrate.
8. A method for identifying a compound that is useful in treating a
neuropsychiatric or neurodevelopmental disorder, which method
comprises detecting modulation of of PAMP expression in a
transgenic animal that expresses PAMP, wherein the animal is
contacted with the compound.
9. The method of claim 8, wherein the disorder is
schizophrenia.
10. (canceled)
11. (canceled)
12. A method for identifying a compound that is useful in treating
a neuropsychiatric or neurodevelopmental disorder, which method
comprises: (a) contacting a complex between a presenilin associated
membrane protein (PAMP) and an agent, which agent provides a
detectable conformational or functional change in said PAMP upon
interaction with a substance being analyzed for activity against a
neurodegenerative disease, with a test compound; and (b) detecting
a conformational or functional change in PAMP in the complex.
13. The method of claim 12, wherein the disorder is
schizophrenia.
14. The method of claim 12, wherein the test compound is a protein
that interacts with PAMP.
15. A method for treating neuropsychiatric or neurodevelopmental
disorder in a mammalian host which expresses at least one PAMP
protein or a naturally occurring variant, which method comprises
administering to the host an amount of compound effective to
modulate PAMP expression in the host.
16. The method of claim 15, wherein the disorder is schizophrenia.
Description
[0001] This patent application claims the priority of U.S.
provisional patent application No. 60/229,889, filed Sep. 1, 2000,
which is incorporated herein by reference in its entirety.
FIELD OF THE INVENTION
[0002] The present invention relates generally to the field of
neurological and physiological dysfunctions associated with
neuropsychiatric and neurodevelopmental diseases, especially
schizophrenia. More particularly, the invention is concerned with
the identification of proteins associated with neuropsychiatric and
neurodevelopmental diseases, especially schizophrenia, and relates
to methods of diagnosing these diseases, and to methods of
screening for candidate compounds which modulate the interaction of
a certain protein, specifically Presenilin Associated Membrane
Protein ("PAMP"), with presenilin proteins.
BACKGROUND OF THE INVENTION
[0003] The origin of and causes for schizophrenia, one of the most
serious neuropsychiatric disorders, have long been sought after. A
number of studies have suggested that schizophrenia is
predominantly genetic, but it has proven difficult to show a
significant genetic linkage. However, recently, a novel locus
associated with inherited susceptibility to schizophrenia has been
mapped to chromosome 1 q21-q22, near the anonymous DNA markers
D1S1653, D1S1679, and D1S1677 (Brzustowicz et al., 2000).
Furthermore, several lines of evidence, both from morphological and
neuropsychological findings, now indicate that schizophrenia may be
a disease of central nervous system development (reviewed in Stefan
et al, 1997). For example, Falkai et al., 2000, provided
quantitative data showing that the positioning of neuron (pre-alpha
cell) clusters was abnormal in schizophrenia patients, supporting
the theory that schizophrenia derives from impaired brain
development. Such abnormal neuron positions could, e.g., arise from
failures of neuronal migration during fetal development.
[0004] An important pathway implicated in the development of the
nervous system, as well as in schizophrenia, is the Notch signaling
pathway. Notch is a protein receptor for inhibitory signals that
shape the pattern of the nervous system, and the localization of
Notch signaling is crucial for determining where neural precursor
cells arise (Baker, 2000). In a series of 80 British
parent-offspring trios, the NOTCH4 locus was highly associated with
schizophrenia (Wei and Hemmings, 2000). Possible candidate sites
conferring susceptibility to schizophrenia included an A-to-G
substitution in the promoter region, and the (CTG).sub.n repeat in
exon 1, of NOTCH4.
[0005] The presenilin proteins, i.e., presenilin 1 (PS1, encoded by
the PS1 gene) and 2 (PS2, encoded by the PS2 gene), are involved in
the Notch pathway, and form a close functional relationship with
Notch during cell fate determination in a variety of species
(Selkoe, 2000). Several lines of evidence have suggested roles for
PS1 and PS2 genes in developmental, apoptotic signaling and in the
regulation of proteolytic cleavage of the .beta.-amyloid precursor
protein (.beta.APP) (Levitan et al., 1995; Wong et al., 1997; Shen
et al., 1997; Wolozin et al., 1996; De Strooper et al., 1998). For
example, the PS1 gene is associated with migration defects in the
central nervous system of PS1-/- mice (Hartmann et al., 1999;
Handler et al., 2000). In addition, a mutation in .beta.APP
(.beta.APP.sub.Ala713Val) has been described in one family with a
schizophrenia-like illness (Jones et al., 1992), further
implicating the PS1/.beta.APP/Notch pathways in schizophrenia and
related disorders. However, just how these putative functions are
mediated, and how they relate to the abnormal metabolism of the
.beta.APP associated with PS1 and PS2 mutations remains to be
elucidated (Martin et al., 1995; Scheuner et al., 1996; Citron et
al., 1997; Duff et al., 1996; Borchelt et al., 1996). The
identification and cloning of normal as well as mutant PS1 and PS2
genes and gene products are described in detail in co-pending
commonly assigned U.S. application Ser. No. 08/431,048, filed Apr.
28, 1995; Ser. No. 08/496,841, filed Jun. 28, 1995; Ser. No.
08/509,359, filed Jul. 31, 1995; and Ser. No. 08/592,541, filed
Jan. 26, 1996, the disclosures of which are incorporated herein by
reference.
[0006] A new protein which specifically interacts with PS1 and PS2
has recently been discovered. This transmembrane protein, herein
referred to as "Presenilin Associated Membrane Protein" or "PAMP",
is expressed in multiple tissues (e.g., brain, kidney, lung, etc.).
PAMP is described in co-pending commonly assigned U.S. application
Ser. No. 09/541,094, filed Mar. 31, 2000, which is specifically
incorporated herein by reference. The PAMP gene and gene product is
implicated in the biochemical pathways affected in Alzheimer's
Disease (AD), and may also have a role in other dementias, amyloid
angiopathies, and developmental disorders such as spina bifida.
Interestingly, the gene associated with inherited susceptibility to
schizophrenia (see Brzustowicz, supra) also contains the PAMP gene
(Yu et al, 2000).
[0007] A need exists for new methods and reagents to more
accurately and effectively diagnose and treat schizophrenia as well
as other neuropsychiatric, neurodevelopmental, and
neurodegenerative diseases. In addition, further insights into PAMP
and its interaction with PS proteins and other components may lead
to new diagnostic and treatment methods for schizophrenia and other
related CNS diseases.
SUMMARY OF THE INVENTION
[0008] The present invention provides new uses of the PAMP gene,
the product of the gene, and mutations and polymorphisms thereof in
the study and treatment of a variety of neurological disorders,
especially schizophrenia. Applicants have surprisingly discovered
that PAMP plays a role in the development of schizophrenia. The
PAMP gene and the product of the PAMP gene therefore present new
therapeutic targets for the treatment of a variety of neurological
disorders, especially schizophrenia. Moreover, the PAMP gene will
be useful for generating animal and cellular models of
schizophrenia.
[0009] Thus, PAMP nucleic acids, proteins and peptides, antibodies
to PAMP, cells transformed with PAMP nucleic acids, and transgenic
animals altered with PAMP nucleic acids that possess various
utilities, are described herein for the diagnosis, therapy and
continued investigation of neuropsychiatric and neurodevelopmental
disorders, especially schizophrenia. Furthermore, mutant PAMP
nucleic acids, proteins, or peptides, cells transfected with
vectors comprising mutant PAMP nucleic acids, transgenic animals
expressing mutant PAMP or peptides thereof, and their use in
studying neuropsychiatric and neurodevelopmental disorders,
especially schizophrenia, or developing improved diagnostic or
therapeutic methods for such disorders, are presented herein.
[0010] The invention provides a method for detecting a mutation in
PAMP associated with neuropsychiatric and neurodevelopmental
disorders, especially schizophrenia, comprising obtaining a nucleic
acid sample from an individual diagnosed with or suspected of
having schizophrenia or another neuropsychiatric or
neurodevelopmental disorder, and sequencing a gene encoding PAMP
from said sample. In particular, such methods can identify normal
human alleles as well as mutant alleles of PAMP genes which are
causative of or contribute to neuropsychiatric or
neurodevelopmental diseases, especially schizophrenia.
[0011] The invention also provides a method for diagnosing
individuals predisposed to or having a neuropsychiatric and/or
neurodevelopmental disorder such as schizophrenia, comprising
obtaining a nucleic acid sample from an individual diagnosed with
or suspected of having such a disorder, and sequencing a gene
encoding PAMP from said sample.
[0012] The invention also provides a method for diagnosing
individuals predisposed to or having a neuropsychiatric and/or
neurodevelopmental disorder such as schizophrenia, comprising
obtaining cells that contain nucleic acid encoding PAMP, and under
non-pathological conditions, transcribing the nucleic acid, and
measuring a level of transcriptional activity of the nucleic
acid.
[0013] The invention further provides a method for diagnosing
individuals predisposed to or having a neuropsychiatric or
neurodevelopmental disorder, especially schizophrenia, comprising
obtaining cells from an individual that express nucleic acid
encoding PAMP, and measuring PAMP activity. Alternatively, PAMP
could be isolated from that individual to investigate, for example,
whether the PAMP amino acid sequence is similar or different from
wild-type PAMP, and/or whether PAMP expression levels differ from
typical PAMP levels. In an alternative embodiment, the activity or
abundance of a PAMP substrate may be measured.
[0014] The invention also provides a method for identifying
putative agents that affect a neuropsychiatric and/or
neurodevelopmental disorder, especially schizophrenia, comprising
administering one or more putative agents to a transgenic animal
and detecting a change in PAMP activity.
[0015] The invention also provides a method for identifying
putative agents that affect a neuropsychiatric and/or
neurodevelopmental disorder, especially schizophrenia, comprising
adding one or more said agents to the reconstituted system
described above, and detecting a change in PAMP activity.
[0016] The invention also provides a method for identifying
putative agents that affect a neuropsychiatric and/or
neurodevelopmental disorder, especially schizophrenia, comprising
adding one or more said agents to the complex described above, and
detecting a conformational change in PAMP.
[0017] The invention also provides a method for identifying
proteins that interact with PAMP, comprising contacting a substance
to the reconstituted system discussed above, and detecting a change
in PAMP activity.
[0018] The invention also provides animal and cellular models of
schizophrenia or related disorders that comprise a PAMP gene as a
therapeutic target for the development of drugs which interact with
PAMP, and thus may be useful in the treatment and prevention of
schizophrenia or related disorders.
[0019] Further the invention provides for a method for identifying
substances that modulate PAMP activity, comprising contacting a
sample containing one or more substances with PAMP, or a PAMP
mutant, or functional fragments thereof, and a PAMP substrate,
measuring PAMP activity, and determining whether a change in PAMP
activity occurs. In a preferred embodiment, the substance is a PAMP
inhibitor. In another preferred embodiment, the substance
stimulates PAMP activity.
[0020] These and other aspects of the invention are further
elaborated in the Detailed Description of the Invention and
Examples, infra.
DESCRIPTION OF THE DRAWINGS
[0021] FIGS. 1A and 1B. Predicted amino acid sequences for human
(SEQ ID NO:14), mouse (SEQ ID NO:16), D. melanogaster (SEQ ID
NO:18) and C. elegans (SEQ ID NO:12) PAMP orthologues.
DETAILED DESCRIPTION OF THE INVENTION
[0022] According to the invention, the PAMP gene and its product,
the PAMP protein, present new therapeutic targets for the treatment
of a variety of neurological disorders, especially schizophrenia.
Provided herein are also new strategies to create animal and
cellular models based on PAMP or PAMP mutants to study
schizophrenia and potential treatment strategies. The invention
also offers the potential for new diagnostic screening methods for
schizophrenia, wherein the PAMP gene and PAMP protein are
investigated.
PAMP
[0023] The invention is based, in part, on the discovery that the
PAMP gene and the PAMP protein play critical roles in schizophrenia
and other neuropsychiatric disorders. PAMP ("Presenilin Associated
Membrane Protein"), is a novel Type I transmembrane protein that is
closely involved in CNS development via its interactions with Notch
processing, PS1, PS2 and with the .alpha.- and .beta.-secretase
derived fragments of .beta.APP. Multiple studies have indicated
that defects in CNS development, such as defects in neuronal
migration, are associated with schizophrenia, and PAMP is linked to
this disorder through several lines of evidence, such as (1) the
PAMP gene maps to the same location as the schizophrenia
susceptibility gene, as described above; (2) PAMP interacts with
PS1, which is associated with migration defects in the central
nervous system in PS1-/- mice (see above); (3) PAMP is involved in
the Notch signaling pathway, one gene locus of which (NOTCH4) is
implicated in schizophrenia (see above); and (4) a mutation in
.beta.APP is associated with a schizophrenia-like illness (see
above). Therefore, PAMP can contribute to the development of
schizophrenia via several routes, e.g., through mutations and/or
polymorphisms in PAMP, variations in its expression levels, and
defects in its interactions with other components in neural
development and/or migration.
[0024] As referred to herein, "PAMP" means a native or mutant
full-length protein, or fragments thereof, that interacts with the
PAMP-interacting domain of a presenilin protein. PAMP is also known
under the name "Nicastrin". Human, murine, D. melanogaster and C.
elegans orthologues are provided.
[0025] Experimental data indicate that PAMP, PS1, and PS2 exist in
the same high molecular weight protein complex, and PAMP and PS1
are both co-localized to intracellular membranes in the endoplasmic
reticulum and Golgi apparatus. Abolition of functional expression
of a C. elegans homologue of this protein leads to the development
of Notch-like developmental defects. This shows that PAMP is also
intimately involved in the processing of not only .beta.APP, but
also other molecules, such as Notch and its homologues. For
example, PAMP can bind to membrane-bound Notch. From expressed
sequence tags (EST) databases, it is apparent that, like PS1 and
PS2, PAMP is expressed in multiple tissues.
[0026] Various structural features characterize PAMP (GenBank
Accession No. Q92542; SEQ ID NO: 14). The nucleotide sequence (SEQ
ID NO: 13) of human PAMP predicts that the gene encodes a Type 1
transmembrane protein of 709 amino acids (SEQ ID NO: 14), the
protein having a short hydrophilic C-terminus (.about.20 residues),
a hydrophobic transmembrane domain (15-20 residues), and a longer
N-terminal hydrophilic domain which contains several potentially
functional sequence motifs as listed below in Table 1. The PAMP
sequence also contains a Trp-Asp (WD) repeat (residue 226), at
least one "DTG" motif (residues 91-93) present in eukaryotic
aspartyl proteases, as well as several "DTA/DTAE" motifs (residues
480-482, 504-506) present in viral aspartyl proteases. There are
also four conserved cysteine residues in the N-terminal hydrophilic
domain (Cys.sub.195, Cys.sub.213, Cys.sub.230, and Cys.sub.248 in
human PAMP) having a periodocity of 16-17 residues, which may form
a functional domain (e.g., a metal binding domain or disulfide
bridge for tertiary structure stabilization). Subdomains of PAMP
have weak homologies to a variety of peptidases. For example,
residues 322-343, 361-405, and 451-466 have 46% (p=0.03) similarity
to another hypothetical protein; C. elegans aminopeptidase
hydrolase precursor signal antigen transmembrane receptor zinc
glycoprotein (SWISS-PROT; see expasy.ch/sprot on the World-Wide Web
(www); Accession No. Q93332). TABLE-US-00001 TABLE 1 Potential
functional sequence motifs in PAMP (SEQ ID NO: 14). Potential
function PAMP residue (amino acid sequence) N-asparaginyl
glycosylation 45 (NKTA), 55 (NATH), 187 (NETK), 200 (NLSQ), 204
(NGSA), 264 (NTTG), 387 (NESV), 417 (NQSQ), 435 (NISG), 464 (NVSY),
506 (NFSD), 530 (NNSW), 562 (NTTY), 573 (NLTG), 580 (NLTR), 612
(NETD) Glycosaminoglycan attachment 404 (SGAG) Myristolation 5
(GGGSGA), 29 (GLCRGN), 61 (GCQSSI), 120 (GLAVSL), 146 (GVYSNS), 167
(GNGLAY), 205 (GSAPTF), 294 (GAESAV), 438 (GVVLAD), 446 (GAFHNK),
504 (GTNFSD), 576 (GTVVNL) Phosphorylation sites for cAMP- and 232
(RRSS) cGMP-dependent protein kinase Phosphorylation sites for
protein 115 (TSR), 268 (TLK), 340 (SSR), 384 (SQK), 389 kinase C
(SVR), 483 (TAK), 614 (TDR), 624 (TAR) Phosphorylation sites for
casein kinase 8 (SGAD), 280 (TRLD), 361 (SFVE), 372 (TSLE), 455 II
(SIYD), 466 (SYPE), 472 (SPEE), 641 (SSTE), 647 (TWTE)
[0027] The invention is further based on the identification of
conserved functional domains, based on comparison and evaluation of
the predicted amino acid sequences of human (SEQ ID NO: 14), murine
(SEQ ID NO: 16), D. melanogaster (SEQ ID NO: 18), and C. elegans
(SEQ ID NO: 12) orthologues of PAMP. "PAMP" can be characterized by
the presence of conserved structural features, relative to
orthologues from D. melanogaster and C. elegans. Nucleotide
sequences encoding homologous hypothetical proteins exist in mice
multiple EST, and C. elegans (GenBank; see ncbi.nlm.nih.gov on the
World-Wide Web (www); Accession No. Z75714; 37% similarity,
p=8.7e.sup.-26) (Wilson et al., 1994). These hypothetical murine
and nematode proteins have a similar topology and contain similar
functional motifs to human PAMP. The existence of such homology
predicts that similar proteins will be detected in other species
including Xenopus, and Zebra fish, to mention a few such
possibilities. By comparing the predicted amino acid sequences of
human (SEQ ID NO: 14), murine (SEQ ID NO: 16), D. melanogaster (SEQ
ID NO: 18), and C. elegans (SEQ ID NO: 12) PAMP proteins, we have
deduced a series of conserved functional domains. One domain has
chemical similarities to cyclic nucleotide binding domains of other
proteins, and may have some regulatory role on a potential complex
formed between PS1:PAMP and the C-terminal fragment of .beta.APP,
derived either from .alpha.- or .beta.-secretase. These putative
functional domains are sites for therapeutic target development by
deploying drugs which might interact with these sites to modulate
.beta.APP processing via this complex.
[0028] The term "PAMP" also refers to functionally active fragments
of the protein. Such fragments include, but are not limited to,
peptides that contain an epitope, e.g., as determined by
conventional algorithms such as hydrophilicity/hydrophobicity
analysis for antibody epitopes, and amphipathicity or consensus
algorithms for T cell epitopes (Spouge et al., 1987; Margalit et
al., 1987; Rothbard, 1986; Rothbard and Taylor, 1988). More
preferably, a functionally active fragment of PAMP is a conserved
domain, relative to the D. melanogaster and C. elegans orthologues.
A specific functionally active fragment of PAMP is a fragment that
interacts with PS1 or PS2, or both.
[0029] PAMP also encompasses naturally occurring variants,
including other mammalian PAMPs (readily identified, as shown
herein for murine PAMP, based on the presence of the structural
features set forth above), allelic variants of PAMP from other
human sources (including variants containing polymorphisms that are
predictive of disease propensity or of response to pharmacological
agents), and mutant forms of PAMP or PAMP genes that are associated
with neurological diseases and disorders (such as spina bifida),
particularly neuropsychiatric disorders (such as schizophrenia).
Also included are artificial PAMP mutants created by standard
techniques such as site directed mutagenesis or chemical
synthesis.
[0030] A PAMP "substrate" may be a polypeptide or protein, or any
other type of compound, with which PAMP interacts physiologically.
Examples of PAMP substrates include PS1, PS2, and .beta.APP.
Furthermore, A PAMP "ligand" may be a polypeptide, protein, lipid,
carbohydrate, vitamin, mineral, amino acid, or any other type of
compound which binds to PAMP Hypothetically, PAMP may function as a
receptor which modulates PS1/PS2/.beta.APP processing in response
to signal (ligand) dependent interactions with PAMP.
Definitions
[0031] In accordance with the present invention there may be
employed conventional molecular biology, microbiology, and
recombinant DNA techniques within the skill of the art. Such
techniques are explained fully in the literature. See, e.g.,
Sambrook, Fritsch & Maniatis, 1989; Glover, 1985; M. J. Gait,
1984; Hames &Higgins, 1985; Hames & S. J. Higgins, 1984;
Freshney, 1986; IRL Press, 1986; Perbal, 1984; Ausubel et al.,
1994.
[0032] If appearing herein, the following terms shall have the
definitions set out below.
[0033] "Neuropsychiatric disorders" or "diseases" include
recognized variants of overt schizophrenia (e.g., paranoid,
catatonic), other related psychoses such as schizoaffective,
schizotypal, schizophreniform and delusional disorders, and
personality disorders such as schizoid personality disorder,
schizotypal personality disorder, and paranoid personality disorder
(see definitions in DSM-III-R, Diagnostic and Statistical Manual of
the American Psychiatric Association; and Flaum et al., 1997).
[0034] The use of italics (e.g., PAMP) indicates a nucleic acid
molecule (cDNA, mRNA, gene, etc.); normal text (e.g., PAMP)
indicates the polypeptide or protein.
[0035] In a specific embodiment, the term "about" or
"approximately" means within 20%, preferably within 10%, and more
preferably within 5% of a given value or range. Alternatively,
particularly in biological systems which are often responsive to
order of magnitude changes, the term about means within an order of
magnitude of a given value, preferably within a multiple of about
5-fold, and more preferably within a factor of about 2-fold of a
given value.
[0036] As used herein, the term "isolated" means that the
referenced material is free of components found in the natural
environment in which the material is normally found. In particular,
isolated biological material is free of cellular components. In the
case of nucleic acid molecules, an isolated nucleic acid includes a
PCR product, an isolated mRNA, a cDNA, or a restriction fragment.
In another embodiment, an isolated nucleic acid is preferably
excised from the chromosome in which it may be found, and more
preferably is no longer joined to non-regulatory, non-coding
regions, or to other genes, located upstream or downstream of the
gene contained by the isolated nucleic acid molecule when found in
the chromosome. In yet another embodiment, the isolated nucleic
acid lacks one or more introns. Isolated nucleic acid molecules can
be inserted into plasmids, cosmids, artificial chromosomes, and the
like. Thus, in a specific embodiment, a recombinant nucleic acid is
an isolated nucleic acid. An isolated protein may be associated
with other proteins or nucleic acids, or both, with which it
associates in the cell, or with cellular membranes if it is a
membrane-associated protein. An isolated organelle, cell, or tissue
is removed from the anatomical site in which it is found in an
organism. An isolated material may be, but need not be,
purified.
[0037] The term "purified" as used herein refers to material that
has been isolated under conditions that reduce or eliminate
unrelated materials, i.e., contaminants. For example, a purified
protein is preferably substantially free of other proteins or
nucleic acids with which it is associated in a cell; a purified
nucleic acid molecule is preferably substantially free of proteins
or other unrelated nucleic acid molecules with which it can be
found within a cell.
[0038] As used herein, the term "substantially free" is used
operationally, in the context of analytical testing of the
material. Preferably, purified material substantially free of
contaminants is at least 50% pure; more preferably, at least 90%
pure, and more preferably still at least 99% pure. Purity can be
evaluated by chromatography, gel electrophoresis, immunoassay,
composition analysis, biological assay, and other methods known in
the art.
[0039] The term "host cell" means any cell of any organism that is
selected, modified, transformed, grown, or used or manipulated in
any way, for the production of a substance by the cell, for example
the expression by the cell of a gene, a DNA or RNA sequence, a
protein or an enzyme. Host cells can further be used for screening
or functional assays, as described infra. A host cell has been
"transfected" by exogenous or heterologous DNA when such DNA has
been introduced inside the cell. A cell has been "transformed" by
exogenous or heterologous DNA when the transfected DNA is expressed
and effects a function or phenotype on the cell in which it is
expressed. The term "expression system" means a host cell
transformed by a compatible expression vector and cultured under
suitable conditions e.g. for the expression of a protein coded for
by foreign DNA carried by the vector and introduced to the host
cell.
[0040] Proteins and polypeptides can be made in the host cell by
expression of recombinant DNA. As used herein, the term
"polypeptide" refers to an amino acid-based polymer, which can be
encoded by a nucleic acid or prepared synthetically. Polypeptides
can be proteins, protein fragments, chimeric proteins, etc.
Generally, the term "protein" refers to a polypeptide expressed
endogenously in a cell, e.g., the naturally occurring form (or
forms) of the amino acid-based polymer.
[0041] A "coding sequence" or a sequence "encoding" an expression
product, such as a RNA, polypeptide, protein, or enzyme, is a
nucleotide sequence that, when expressed, results in the production
of that RNA, polypeptide, protein, or enzyme, i.e., the nucleotide
sequence encodes an amino acid sequence for that polypeptide,
protein or enzyme. A coding sequence for a protein may include a
start codon (usually ATG) and a stop codon.
[0042] The coding sequences herein may be flanked by natural
regulatory (expression control) sequences, or may be associated
with heterologous sequences, including promoters, internal ribosome
entry sites (IRES) and other ribosome binding site sequences,
enhancers, response elements, suppressors, signal sequences,
polyadenylation sequences, introns, 5'- and 3'-non-coding regions,
and the like. The nucleic acids may also be modified by many means
known in the art. Non-limiting examples of such modifications
include methylation, "caps", substitution of one or more of the
naturally occurring nucleotides with an analog, and internucleotide
modifications.
[0043] The term "gene", also called a "structural gene" means a DNA
sequence that codes for or corresponds to a particular sequence of
ribonucleic acids or amino acids which comprise all or part of one
or more proteins, and may or may not include regulatory DNA
sequences, such as promoter sequences, which determine for example
the conditions under which the gene is expressed.
[0044] A "promoter sequence" is a DNA regulatory region capable of
binding RNA polymerase in a cell and initiating transcription of a
downstream (3' direction) coding sequence. For purposes of defining
the present invention, the promoter sequence is bounded at its 3'
terminus by the transcription initiation site and extends upstream
(5' direction) to include the minimum number of bases or elements
necessary to initiate transcription at levels detectable above
background.
[0045] A coding sequence is "under the control" or "operatively
associated with" transcriptional and translational control
sequences in a cell when RNA polymerase transcribes the coding
sequence into mRNA, which then may be trans-RNA spliced (if it
contains introns) and translated into the protein encoded by the
coding sequence.
[0046] The terms "express" and "expression" mean allowing or
causing the information in a gene or DNA sequence to become
manifest, for example producing a protein by activating the
cellular functions involved in transcription and translation of a
corresponding gene or DNA sequence. A DNA sequence is expressed in
or by a cell to form an "expression product" such as a protein. The
expression product itself, e.g. the resulting protein, may also be
said to be "expressed" by the cell.
[0047] The term "transfection" means the introduction of a foreign
nucleic acid into a cell. The term "transformation" means the
introduction of a "foreign" (i.e. extrinsic or extracellular) gene,
DNA or RNA sequence to a host cell, so that the host cell will
express the introduced gene or sequence to produce a desired
substance, typically a protein or enzyme coded by the introduced
gene or sequence. The introduced gene or sequence may also be
called a "cloned", "foreign", or "heterologous" gene or sequence,
and may include regulatory or control sequences used by a cell's
genetic machinery. The gene or sequence may include nonfunctional
sequences or sequences with no known function. A host cell that
receives and expresses introduced DNA or RNA has been "transformed"
and is a "transformant" or a "clone." The DNA or RNA introduced to
a host cell can come from any source, including cells of the same
genus or species as the host cell, or cells of a different genus or
species.
[0048] The terms "vector", "cloning vector" and "expression vector"
mean the vehicle by which a DNA or RNA sequence (e.g., a foreign
gene) can be introduced into a host cell, so as to transform the
host and promote expression (e.g., transcription and translation)
of the introduced sequence. Vectors include plasmids, phages,
viruses, etc. A "cassette" refers to a DNA coding sequence or
segment of DNA that codes for an expression product that can be
inserted into a vector at defined restriction sites. The cassette
restriction sites are designed to ensure insertion of the cassette
in the proper reading frame. Generally, foreign DNA is inserted at
one or more restriction sites of the vector DNA, and then is
carried by the vector into a host cell along with the transmissible
vector DNA. A segment or sequence of DNA having inserted or added
DNA, such as an expression vector, can also be called a "DNA
construct." Recombinant cloning vectors will often include one or
more replication systems for cloning or expression, one or more
markers for selection in the host, e.g. antibiotic resistance, and
one or more expression cassettes.
[0049] A "knockout mammal" is a mammal (e.g., mouse) that contains
within its genome a specific gene that has been inactivated by the
method of gene targeting (see, e.g., U.S. Pat. No. 5,777,195 and
U.S. Pat. No. 5,616,491). A knockout mammal includes both a
heterozygote knockout (i.e., one defective allele and one wild-type
allele) and a homozygous mutant. Preparation of a knockout mammal
requires first introducing a nucleic acid construct that will be
used to suppress expression of a particular gene into an
undifferentiated cell type termed an embryonic stem cell. This cell
is then injected into a mammalian embryo. A mammalian embryo with
an integrated cell is then implanted into a foster mother for the
duration of gestation. Zhou, et al., 1995 describes PPCA knock-out
mice. Knockout mice can be used to study defects in neurological
development or neurodegenerative diseases. Disease phenotypes that
develop can provide a platform for further drug discovery.
[0050] The term "knockout" refers to partial or complete
suppression of the expression of at least a portion of a protein
encoded by an endogenous DNA sequence in a cell. The term "knockout
construct" refers to a nucleic acid sequence that is designed to
decrease or suppress expression of a protein encoded by endogenous
DNA sequences in a cell. The nucleic acid sequence used as the
knockout construct is typically comprised of (1) DNA from some
portion of the gene (exon sequence, intron sequence, and/or
promoter sequence) to be suppressed and (2) a marker sequence used
to detect the presence of the knockout construct in the cell. The
knockout construct is inserted into a cell, and integrates with the
genomic DNA of the cell in such a position so as to prevent or
interrupt transcription of the native DNA sequence. Such insertion
usually occurs by homologous recombination (i.e., regions of the
knockout construct that are homologous to endogenous DNA sequences
hybridize to each other when the knockout construct is inserted
into the cell and recombine so that the knockout construct is
incorporated into the corresponding position of the endogenous
DNA). The knockout construct nucleic acid sequence may comprise 1)
a full or partial sequence of one or more exons and/or introns of
the gene to be suppressed, 2) a full or partial promoter sequence
of the gene to be suppressed, or 3) combinations thereof.
Typically, the knockout construct is inserted into an embryonic
stem cell (ES cell) and is integrated into the ES cell genomic DNA,
usually by the process of homologous recombination. This ES cell is
then injected into, and integrates with, the developing embryo.
[0051] Generally, for homologous recombination, the DNA will be at
least about 1 kilobase (kb) in length and preferably 3-4 kb in
length, thereby providing sufficient complementary sequence for
recombination when the knockout construct is introduced into the
genomic DNA of the ES cell.
[0052] A "knock-in" mammal is a mammal in which an endogenous gene
is substituted with a heterologous gene or a modified variant of
the endogenous gene (Roemer et al., 1991). Preferably, the
heterologous gene is "knocked-in" to a locus of interest, for
example into a gene that is the subject of evaluation of expression
or function, thereby linking the heterologous gene expression to
transcription from the appropriate promoter (in which case the gene
may be a reporter gene; see Elefanty et al., 1998). This can be
achieved by homologous recombination, transposon (Westphal and
Leder, 1997), using mutant recombination sites (Araki et al., 1997)
or PCR (Zhang and Henderson, 1998).
[0053] The phrases "disruption of the gene" and "gene disruption"
refer to insertion of a nucleic acid sequence into one region of
the native DNA sequence (usually one or more exons) and/or the
promoter region of a gene so as to decrease or prevent expression
of that gene in the cell as compared to the wild-type or naturally
occurring sequence of the gene. By way of example, a nucleic acid
construct can be prepared containing a DNA sequence encoding an
antibiotic resistance gene which is inserted into the DNA sequence
that is complementary to the DNA sequence (promoter and/or coding
region) to be disrupted. When this nucleic acid construct is then
transfected into a cell, the construct will integrate into the
genomic DNA. Thus, some progeny of the cell will no longer express
the gene, or will express it at a decreased level, as the DNA is
now disrupted by the antibiotic resistance gene.
[0054] The term "heterologous" refers to a combination of elements
not naturally occurring. For example, heterologous DNA refers to
DNA not naturally located in the cell, or in a chromosomal site of
the cell. Preferably, the heterologous DNA includes a gene foreign
to the cell. A heterologous expression regulatory element is a such
an element operatively associated with a different gene than the
one it is operatively associated with in nature. In the context of
the present invention, an gene is heterologous to the recombinant
vector DNA in which it is inserted for cloning or expression, and
it is heterologous to a host cell containing such a vector, in
which it is expressed, e.g., a CHO cell.
[0055] The terms "mutant" and "mutation" mean any detectable change
in genetic material, e.g. DNA, or any process, mechanism, or result
of such a change. This includes gene mutations, in which the
structure (e.g., DNA sequence) of a gene is altered, any gene or
DNA arising from any mutation process, and any expression product
(e.g., protein) expressed by a modified gene or DNA sequence. The
term "variant" may also be used to indicate a modified or altered
gene, DNA sequence, enzyme, cell, etc., i.e., any kind of
mutant.
[0056] "Sequence-conservative variants" of a polynucleotide
sequence are those in which a change of one or more nucleotides in
a given codon position results in no alteration in the amino acid
encoded at that position.
[0057] "Function-conservative variants" are those in which a given
amino acid residue in a protein or enzyme has been changed without
altering the overall conformation and function of the polypeptide,
including, but not limited to, replacement of an amino acid with
one having similar properties (such as, for example, polarity,
hydrogen bonding potential, acidic, basic, hydrophobic, aromatic,
and the like). Amino acids with similar properties are well known
in the art. For example, arginine, histidine and lysine are
hydrophilic-basic amino acids and may be interchangeable.
Similarly, isoleucine, a hydrophobic amino acid, may be replaced
with leucine, methionine or valine. Such changes are expected to
have little or no effect on the apparent molecular weight or
isoelectric point of the protein or polypeptide. Amino acids other
than those indicated as conserved may differ in a protein or enzyme
so that the percent protein or amino acid sequence similarity
between any two proteins of similar function may vary and may be,
for example, from 70% to 99% as determined according to an
alignment scheme such as by the Cluster Method, wherein similarity
is based on the MEGALIGN algorithm. A "function-conservative
variant" also includes a polypeptide or enzyme which has at least
60% amino acid identity as determined by BLAST (Altschul, et al.,
1990) or FASTA algorithms, preferably at least 75%, most preferably
at least 85%, and even more preferably at least 90%, and which has
the same or substantially similar properties or functions as the
native or parent protein or enzyme to which it is compared.
[0058] An "ortholog" to a protein means a corresponding protein
from another species. Orthologous proteins typically have similar
functions in different species, and can also be substantially
homologous.
[0059] As used herein, the term "homologous" in all its grammatical
forms and spelling variations refers to the relationship between
proteins that possess a "common evolutionary origin," including
proteins from superfamilies (e.g., the immunoglobulin superfamily)
and homologous proteins from different species (e.g., myosin light
chain, etc.) (Reeck et al., 1987). Such proteins (and their
encoding genes) have sequence homology, as reflected by their
sequence similarity, whether in terms of percent similarity or the
presence of specific residues or motifs. Motif analysis can be
performed using, for example, the program BLOCKS (blocks.fhcrc.org
on the World-Wide Web).
[0060] Accordingly, the term "sequence similarity" in all its
grammatical forms refers to the degree of identity or
correspondence between nucleic acid or amino acid sequences of
proteins that may or may not share a common evolutionary origin
(see Reeck et al., supra). However, in common usage and in the
instant application, the term "homologous," when modified with an
adverb such as "highly," may refer to sequence similarity and may
or may not relate to a common evolutionary origin.
[0061] In a specific embodiment, two DNA sequences are
"substantially homologous" or "substantially similar" when at least
about 80%, and most preferably at least about 90 or 95% of the
nucleotides match over the defined length of the DNA sequences, as
determined by sequence comparison algorithms, such as BLAST, FASTA,
DNA Strider, etc. Sequences that are substantially homologous can
be identified by comparing the sequences using standard software
available in sequence data banks, or in a Southern hybridization
experiment under, for example, stringent conditions as defined for
that particular system.
[0062] Similarly, in a particular embodiment, two amino acid
sequences are "substantially homologous" or "substantially similar"
when greater than 80% of the amino acids are identical, or greater
than about 90% are similar (functionally identical). Preferably,
the similar or homologous sequences are identified by alignment
using, for example, the GCG (Genetics Computer Group, Program
Manual for the GCG Package, Version 7, Madison, Wis.) pileup
program, ProteinPredict (dodo.cmpc.columbia.edu/predictprotein on
the World-Wide Web), or any of the programs described above (BLAST,
FASTA, etc.).
[0063] A nucleic acid molecule is "hybridizable" to another nucleic
acid molecule, such as a cDNA, genomic DNA, or RNA, when a single
stranded form of the nucleic acid molecule can anneal to the other
nucleic acid molecule under the appropriate conditions of
temperature and solution ionic strength (see Sambrook et al.,
supra). The conditions of temperature and ionic strength determine
the "stringency" of the hybridization. For preliminary screening
for homologous nucleic acids, low stringency hybridization
conditions, corresponding to a T.sub.m (melting temperature) of
55.degree. C., can be used. Moderate stringency hybridization
conditions correspond to a higher T.sub.m and high stringency
hybridization conditions correspond to the highest T.sub.m.
Hybridization requires that the two nucleic acids contain
complementary sequences, although depending on the stringency of
the hybridization, mismatches between bases are possible. The
appropriate stringency for hybridizing nucleic acids depends on the
length of the nucleic acids and the degree of complementation,
variables well known in the art. The greater the degree of
similarity or homology between two nucleotide sequences, the
greater the value of T.sub.m for hybrids of nucleic acids having
those sequences. The relative stability (corresponding to higher
T.sub.m) of nucleic acid hybridizations decreases in the following
order: RNA:RNA, DNA:RNA, DNA:DNA. For hybrids of greater than 100
nucleotides in length, equations for calculating T.sub.m have been
derived (see Sambrook et al., supra, 9.50-9.51). For hybridization
with shorter nucleic acids, i.e., oligonucleotides, the position of
mismatches becomes more important, and the length of the
oligonucleotide determines its specificity (see Sambrook et al.,
supra, 11.7-11.8). A minimum length for a hybridizable nucleic acid
is at least about 10 nucleotides; preferably at least about 15
nucleotides; and more preferably the length is at least about 20
nucleotides.
[0064] The present invention provides antisense nucleic acids
(including ribozymes), which may be used to inhibit expression of
PAMP, e.g., to disrupt a cellular process (such disruption can be
used in an animal model or therapeutically). An "antisense nucleic
acid" is a single stranded nucleic acid molecule which, on
hybridizing under cytoplasmic conditions with complementary bases
in an RNA or DNA molecule, inhibits the latter's role. If the RNA
is a messenger RNA transcript, the antisense nucleic acid is a
counter transcript or mRNA-interfering complementary nucleic acid.
As presently used, "antisense" broadly includes RNA-RNA
interactions, RNA-DNA interactions, ribozymes and RNase-H mediated
arrest. Antisense nucleic acid molecules can be encoded by a
recombinant gene for expression in a cell (e.g., U.S. Pat. No.
5,814,500; U.S. Pat. No. 5,811,234), or alternatively they can be
prepared synthetically (e.g., U.S. Pat. No. 5,780,607).
[0065] As used herein, the term "oligonucleotide" refers to a
nucleic acid, generally of at least 10, preferably at least 15, and
more preferably at least 20 nucleotides, preferably no more than
100 nucleotides, that is hybridizable to a genomic DNA molecule, a
cDNA molecule, or an mRNA molecule encoding a gene, mRNA, cDNA, or
other nucleic acid of interest. Oligonucleotides can be labeled,
e.g., with .sup.32P-nucleotides or nucleotides to which a label,
such as biotin, has been covalently conjugated. In one embodiment,
a labeled oligonucleotide can be used as a probe to detect the
presence of a nucleic acid. In another embodiment, oligonucleotides
(one or both of which may be labeled) can be used as PCR primers,
e.g., for cloning full length or a fragment of a protein or
polypeptide. In a further embodiment, an oligonucleotide of the
invention can form a triple helix with a nucleic acid (genomic DNA
or mRNA) encoding a protein or polypeptide. Generally,
oligonucleotides are prepared synthetically, preferably on a
nucleic acid synthesizer. Accordingly, oligonucleotides can be
prepared with non-naturally occurring phosphoester analog bonds,
such as thioester bonds, etc. Furthermore, the oligonucleotides
herein may also be modified with a label capable of providing a
detectable signal, either directly or indirectly. Exemplary labels
include radioisotopes, fluorescent molecules, biotin, and the
like.
[0066] Specific non-limiting examples of synthetic oligonucleotides
envisioned for this invention include oligonucleotides that contain
phosphorothioates, phosphotriesters, methyl phosphonates, short
chain alkyl, or cycloalkl intersugar linkages or short chain
heteroatomic or heterocyclic intersugar linkages. Most preferred
are those with CH.sub.2--NH--O--CH.sub.2,
CH.sub.2--N(CH.sub.3)--O--CH.sub.2,
CH.sub.2--O--N(CH.sub.3)--CH.sub.2,
CH.sub.2--N(CH.sub.3)--N(CH.sub.3)--CH.sub.2 and
O--N(CH.sub.3)--CH.sub.2--CH.sub.2 backbones (where phosphodiester
is O--PO.sub.2--O--CH.sub.2). U.S. Pat. No. 5,677,437 describes
heteroaromatic olignucleoside linkages. Nitrogen linkers or groups
containing nitrogen can also be used to prepare oligonucleotide
mimics (U.S. Pat. No. 5,792,844 and U.S. Pat. No. 5,783,682). U.S.
Pat. No. 5,637,684 describes phosphoramidate and
phosphorothioamidate oligomeric compounds. Also envisioned are
oligonucleotides having morpholino backbone structures (U.S. Pat.
No. 5,034,506). In other embodiments, such as the peptide-nucleic
acid (PNA) backbone, the phosphodiester backbone of the
oligonucleotide may be replaced with a polyamide backbone, the
bases being bound directly or indirectly to the aza nitrogen atoms
of the polyamide backbone (Nielsen et al., 1991). Other synthetic
oligonucleotides may contain substituted sugar moieties comprising
one of the following at the 2' position: OH, SH, SCH.sub.3, F, OCN,
O(CH.sub.2).sub.nNH.sub.2 or O(CH.sub.2).sub.nCH.sub.3 where n is
from 1 to about 10; C.sub.1 to C.sub.10 lower alkyl, substituted
lower alkyl, alkaryl or aralkyl; Cl; Br; CN; CF.sub.3; OCF.sub.3;
O-; S-, or N-alkyl; O-, S-, or N-alkenyl; SOCH.sub.3;
SO.sub.2CH.sub.3; ONO.sub.2; NO.sub.2; N.sub.3; NH.sub.2;
heterocycloalkyl; heterocycloalkaryl; aminoalkylamino;
polyalkylamino; substituted silyl; a fluorescein moiety; an RNA
cleaving group; a reporter group; an intercalator; a group for
improving the pharmacokinetic properties of an oligonucleotide; or
a group for improving the pharmacodynamic properties of an
oligonucleotide, and other substituents having similar properties.
Oligonucleotides may also have sugar mimetics such as cyclobutyls
or other carbocyclics in place of the pentofuranosyl group.
Nucleotide units having nucleosides other than adenosine, cytidine,
guanosine, thymidine and uridine, such as inosine, may be used in
an oligonucleotide molecule.
Presenilins
[0067] The presenilin genes (PS1-PS1 and PS2-PS2) encode homologous
polytopic transmembrane proteins that are expressed at low levels
in intracellular membranes including the nuclear envelope, the
endoplasmic reticulum, the Golgi apparatus and some as yet
uncharacterized intracytoplasmic vesicles in many different cell
types including neuronal and non-neuronal cells (see U.S.
application Ser. No. 08/431,048, filed Apr. 28, 1995; Ser. No.
08/496,841, filed Jun. 28, 1995; and Ser. No. 08/509,359, filed
Jul. 31, 1995; PCT Publication No. WO 96/34099, and U.S. Pat. Nos.
5,986,054, 5,040,540, and 6,020,143, the disclosures of which are
specifically incorporated herein by reference; Sherrington et al.,
1995; Rogaev et al., 1995; Levy-Lahad et al., 1995; Doan et al.,
1996; Walter et al., 1996; De Strooper et al., 1997; Lehmann et
al., 1997; Li et al., 1997). Structural studies predict that the
presenilins contain between six and eight transmembrane (TM)
domains organized such that the N-terminus, the C-terminus, and a
large hydrophilic loop following the sixth TM domain are located in
the cytoplasm or nucleoplasm, while the hydrophilic loop between
TM1 and TM2 is located within the lumen of membranous intracellular
organelles (Doan et al., 1996; De Strooper et al., 1997; Lehmann et
al., 1997).
Presenilin Interacting Proteins
[0068] Proteins that interact with the presenilins, i.e.,
PS-interacting proteins, include PAMP, the S5a subunit of the 26S
proteasome (GenBank; Accession No. U51007), Rab11 (GenBank;
Accession Nos. X56740 and X53143), retinoid X receptor B, also
known as nuclear receptor co-regulator or MHC (GenBank Accession
Nos. M84820, and X63522), GT24 (GenBank Accession No. U81004),
.beta.-catenin (Zhou et al., 1997, and Yu et al., supra) as well as
armadillo proteins. These and other PS1 binding proteins are
described in Applicants' copending commonly assigned U.S.
application Ser. No. 08/888,077, filed Jul. 3, 1997, as well as
U.S. application Ser. No. 08/592,541, filed Jan. 26, 1996, and U.S.
application Ser. No. 09/541,094, filed Mar. 31, 2000, the
disclosures of which are incorporated herein by reference.
[0069] PS1 and PS2 interact specifically with at least two members
of the armadillo family of proteins; neuronal plakophilin-related
armadillo protein (Paffenholtz et al., 1997; Paffenholtz et al.,
1999; Zhou et al. (2), 1997) and .beta.-catenin, that are expressed
in both embryonic and post-natal tissues. Moreover, the domains of
PS1 and PS2 that interact with these proteins have been identified.
Mutations in PS1 and PS2 affect the translocation of .beta.-catenin
into the nucleus of both native cells and cells transfected with a
mutant PS gene. These interactions and effects are described in
detail in co-pending commonly assigned U.S. application Ser. No.
09/227,725, filed Jan. 8, 1999, the disclosure of which is
incorporated herein by reference.
[0070] The methods of the present invention are not limited to
mutant presenilins wherein the PAMP-interacting domain is mutated
relative to the wild-type protein. For example, Applicants have
observed that mutations in PS1 (e.g., M146L) outside of the
interacting domain (loop) also affect .beta.-catenin translocation.
These mutations probably disturb the presenilin armadillo
interactions by altering the function of a high MW complex which
contains, e.g., the presenilin and armadillo proteins, as described
in Yu et al., 1998. Moreover, a comparison of the human PS1 (hPS1)
and PS2 (hPS2) sequences reveals that these pathogenic mutations
are in regions of the PS1 protein which are conserved in the PS2
protein. Therefore, corresponding mutations in corresponding
regions of PS2 may also be expected to be pathogenic and are useful
in the methods described herein.
PAMP Mutants
[0071] Mutant PS1 and PS2 genes, and their corresponding amino acid
sequences are described in Applicants' co-pending U.S. application
Ser. No. 08/888,077, filed Jul. 3, 1997, and incorporated herein by
reference. Examples of PS1 mutations include I143T, M146L, L171P,
F177S, A260V, C263R, P264L, P267S, E280A, E280G, A285V, L286V,
.DELTA.291-319, L322V, G384A, L392V, C410Y and I439V. Examples of
PS2 mutations include N141I, M239V and I420T.
[0072] PAMP mutants may cause biochemical changes similar to those
affecting the onset or progression of schizophrenia. Therefore,
artificial PAMP mutations can potentially be used to generate
cellular and other model systems to design treatments and
preventive strategies for schizophrenia and related disorders. Such
mutations may also be used for evaluating whether PAMP is involved
in the pathogenesis of schizophrenia. Since the amyloid-.beta.
(A.beta.) inducing mutations are found in amino acid residues of a
soluble (non-membrane spanning) domain of PAMP, analysis of the
normal structure of this domain and the effects of these and other
nearby mutations on the structure of this domain (and the other
domains of PAMP) provide information for the design of specific
molecular therapeutics.
[0073] In general, modifications of the sequences encoding the
polypeptides described herein may be readily accomplished by
standard techniques such as chemical syntheses and site-directed
mutagenesis. See Gillman et al., 1979; Roberts et al., 1987; and
Innis, 1990. Most modifications are evaluated by routine screening
via an assay designed to select for the desired property.
Antibodies to PAMP
[0074] According to the invention, PAMP polypeptides produced
recombinantly or by chemical synthesis, and fragments or other
derivatives or analogs thereof, including fusion proteins and PAMP
mutants, may be used as an immunogen to generate antibodies that
recognize the PAMP polypeptide. Such antibodies include but are not
limited to polyclonal, monoclonal, chimeric, single chain, Fab
fragments, and an Fab expression library. Such an antibody is
preferably specific for human PAMP, PAMP originating from other
species, or for post-translationally modified (e.g. phosphorylated,
glycosylated) PAMP.
[0075] Various procedures known in the art may be used for the
production of polyclonal antibodies to PAMP polypeptide or
derivative or analog thereof. For the production of antibody,
various host animals can be immunized by injection with the PAMP
polypeptide, or a derivative (e.g., fragment or fusion protein)
thereof, including but not limited to rabbits, mice, rats, sheep,
goats, etc. In one embodiment, the PAMP polypeptide or fragment
thereof can be conjugated to an immunogenic carrier, e.g., bovine
serum albumin (BSA) or keyhole limpet hemocyanin (KLH). Various
adjuvants may be used to increase the immunological response,
depending on the host species, including but not limited to
Freund's (complete and incomplete), mineral gels such as aluminum
hydroxide, surface active substances such as lysolecithin, pluronic
polyols, polyanions, peptides, oil emulsions, keyhole limpet
hemocyanins, dinitrophenol, and potentially useful human adjuvants
such as BCG (bacille Calmette-Guerin) and Corynebacterium parvum.
Antisera may be collected at a chosen time point after
immunization, and purified as desired.
[0076] For preparation of monoclonal antibodies directed toward the
PAMP polypeptide, or fragment, analog, or derivative thereof, any
technique that provides for the production of antibody molecules by
continuous cell lines in culture may be used. These include but are
not limited to the hybridoma technique originally developed by
Kohler and Milstein, 1975, as well as the trioma technique, the
human B-cell hybridoma technique (Kozbor et al., 1983; Cote et al.,
1983), and the EBV-hybridoma technique to produce human monoclonal
antibodies (Cole et al., 1985). Production of human antibodies by
CDR grafting is described in U.S. Pat. Nos. 5,585,089, 5,693,761,
and 5,693,762 to Queen et al., and also in U.S. Pat. No. 5,225,539
to Winter and International Patent Application PCT/WO91/09967 by
Adau et al. In an additional embodiment of the invention,
monoclonal antibodies can be produced in germ-free animals
(International Patent Publication No. WO 89/12690, published 28
Dec. 1989). In fact, according to the invention, techniques
developed for the production of "chimeric antibodies" (Morrison et
al., 1984); Neuberger et al., 1984; Takeda et al., 1985) by
splicing the genes from a mouse antibody molecule specific for an
PAMP polypeptide together with genes from a human antibody molecule
of appropriate biological activity can be used; such antibodies are
within the scope of this invention. Such human or humanized
chimeric antibodies are preferred for use in therapy of human
diseases or disorders (described infra), since the human or
humanized antibodies are much less likely than xenogenic antibodies
to induce an immune response, in particular an allergic response,
themselves.
[0077] According to the invention, techniques described for the
production of single chain antibodies (U.S. Pat. Nos. 5,476,786 and
5,132,405 to Huston; U.S. Pat. No. 4,946,778) can be adapted to
produce PAMP polypeptide-specific single chain antibodies. An
additional embodiment of the invention utilizes the techniques
described for the construction of Fab expression libraries (Huse et
al., 1989) to allow rapid and easy identification of monoclonal Fab
fragments with the desired specificity for an PAMP polypeptide, or
its derivatives, or analogs.
[0078] Antibody fragments which contain the idiotype of the
antibody molecule can be generated by known techniques. For
example, such fragments include but are not limited to: the
F(ab').sub.2 fragment which can be produced by pepsin digestion of
the antibody molecule; the Fab' fragments which can be generated by
reducing the disulfide bridges of the F(ab').sub.2 fragment, and
the Fab fragments which can be generated by treating the antibody
molecule with papain and a reducing agent.
[0079] In the production of antibodies, screening for the desired
antibody can be accomplished by techniques known in the art, e.g.,
radioimmunoassay, ELISA (enzyme-linked immunosorbent assay),
"sandwich" immunoassays, immunoradiometric assays, gel diffusion
precipitin reactions, immunodiffusion assays, in situ immunoassays
(using colloidal gold, enzyme or radioisotope labels, for example),
western blots, precipitation reactions, agglutination assays (e.g.,
gel agglutination assays, hemagglutination assays), complement
fixation assays, immunofluorescence assays, protein A assays, and
immunoelectrophoresis assays, etc. In one embodiment, antibody
binding is detected by detecting a label on the primary antibody.
In another embodiment, the primary antibody is detected by
detecting binding of a secondary antibody or reagent to the primary
antibody. In a further embodiment, the secondary antibody is
labeled. Many means are known in the art for detecting binding in
an immunoassay and are within the scope of the present invention.
For example, to select antibodies which recognize a specific
epitope of an PAMP polypeptide, one may assay generated hybridomas
for a product which binds to an PAMP polypeptide fragment
containing such epitope. For selection of an antibody specific to
an PAMP polypeptide from a particular species of animal, one can
select on the basis of positive binding with PAMP polypeptide
expressed by or isolated from cells of that species of animal.
[0080] The foregoing antibodies can be used in methods known in the
art relating to the localization and activity of the PAMP
polypeptide, e.g., for Western blotting, imaging PAMP polypeptide
in situ, measuring levels thereof in appropriate physiological
samples, etc. using any of the detection techniques mentioned above
or known in the art. Such antibodies can be used to identify
proteins that interact with PAMP, and to detect conformational or
structural changes in PAMP.
[0081] In a specific embodiment, antibodies that agonize or
antagonize the activity of PAMP polypeptide can be generated. They
can also be used to regulate or inhibit PAMP activity
intracellular, i.e., the invention contemplates an intracellular
antibody (intrabody), e.g., single chain Fv antibodies (see
generally, Chen, 1997; Spitz et al., 1996; Indolfi et al., 1996;
Kijima et al., 1995).
PAMP Diagnostic Assays
[0082] The nucleotide sequence and the protein sequence and the
putative biological activity of PAMP or PAMP mutants can all be
used for the purposes of diagnosis of individuals who are at-risk
for, or who actually have, a variety of neurodegenerative diseases
(including Alzheimer's disease, Lewy body variant, Parkinson's
disease-dementia complex, amyotrophic lateral sclerosis etc.),
neuropsychiatric diseases (schizophrenia, depression, mild
cognitive impairment, benign senescent forgetfulness,
age-associated memory loss, etc.), neurodevelopmental disorders
associated with defects in intracellular signal transduction
mediated by factors such as Notch, Delta, Wingless, etc., and
neoplasms (e.g. bowel cancer, etc.) associated with abnormalities
of PS1/PAMP/PS2 mediated regulation of cell death pathways. These
diagnostic entities can be used by searching for alterations in:
the nucleotide sequence of PAMP; in the transcriptional activity of
PAMP; in the protein level as monitored by immunological means
(e.g., ELISA and Western blots); in the amino acid sequence (as
ascertained by Western blotting, amino acid sequence analysis, mass
spectroscopy); and/or in the biological activity of the PAMP
protein as measured by either in vivo methods (e.g., monitoring
.beta.APP processing and the production of amyloid-.beta. peptide
(A.beta.), or other suitable protein substrates for PAMP including
Notch, etc.), or by in vitro assays (using either whole cell or
cell-free assays to measure processing of suitable substrates
including .beta.APP or parts thereof, and other proteins such as
Notch). Any of these assays can also be performed in a transgenic
animal model as well, e.g., to measure the effect of a drug or a
mutation or overexpression of a different gene in vivo.
PAMP Screening Assays
[0083] Identification and isolation of PAMP provides for
development of screening assays, particularly for high throughput
screening of molecules that up- or down-regulate the activity of
PAMP, e.g., by permitting expression of PAMP in quantities greater
than can be isolated from natural sources, or in indicator cells
that are specially engineered to indicate the activity of PAMP
expressed after transfection or transformation of the cells. Any
screening technique known in the art can be used to screen for PAMP
agonists or antagonists. The present invention contemplates screens
for small molecule ligands or ligand analogs and mimics, as well as
screens for natural ligands that bind to and agonize or antagonize
the activity of PAMP in vivo. For example, natural products
libraries can be screened using assays of the invention for
molecules that agonize or antagonize PAMP activity.
[0084] Another approach uses recombinant bacteriophage to produce
large libraries. Using the "phage method" (Scott and Smith, 1990;
Cwirla, et al., 1990; Devlin et al., 1990), very large libraries
can be constructed (10.sup.6-10.sup.8 chemical entities). A second
approach uses primarily chemical methods, of which the Geysen
method (Geysen et al., 1986; Geysen et al., 1987; and the method of
Fodor et al. (1991) are examples. Furka et al., 1988, Furka, 1991,
Houghton (U.S. Pat. No. 4,631,211) and Rutter et al. (U.S. Pat. No.
5,010,175) describe methods to produce a mixture of peptides that
can be tested as agonists or antagonists.
[0085] In another aspect, synthetic libraries (Needels et al.,
1993; Ohlmeyer et al., 1993; Lam et al., WO 92/00252; Kocis et al.,
WO 9428028) and the like can be used to screen for PAMP ligands
according to the present invention.
[0086] Knowledge of the primary sequence of the protein, and the
similarity of that sequence with proteins of known function, can
provide an initial clue as to the inhibitors or antagonists of the
protein. As noted above, identification and screening of
antagonists is further facilitated by determining structural
features of the protein, e.g., using X-ray crystallography, neutron
diffraction, nuclear magnetic resonance spectrometry, and other
techniques for structure determination. These techniques provide
for the rational design or identification of agonists and
antagonists.
[0087] The PAMP protein sequence (including parts thereof) can be
used to deduce the structural organization and topology of PAMP
through the use of a variety of techniques including CD
spectroscopy, nuclear magnetic resonance (NMR) spectroscopy, X-ray
crystallography, and molecular modeling. Sequences for PAMP or PAMP
mutants can also be used to identify proteins which interact with
PAMP either in concert with PS1 and PS2, or independently, using a
variety of methods including co-immunoprecipitation, yeast two
hybrid interaction trap assays, yeast three hybrid interaction trap
assays, chemical cross-linking and co-precipitation studies, etc.
These and other methods are described more fully in co-pending and
commonly assigned U.S. application Ser. No. 08/888,077, filed Jul.
3, 1997, and Ser. No. 09/227,725, filed Jan. 8, 1999, both of which
are incorporated herein by reference. Identification of these
interacting partners will then lead to their use to further
delineate the biochemical pathways leading to the above-mentioned
diseases.
[0088] Finally, the structural analysis of PAMP, when combined with
structural analysis of PS1 and PS2, and other proteins which
interact with PAMP or PAMP mutants, will identify the structural
domains that mediate interactions between these molecules and which
also confer biological activity on PAMP (or PAMP and these other
molecules). These structural domains, and other functional domains,
which can modulate the activity of these structural domains, can
all be modified through a variety of means, including but not
limited to site-directed mutagenesis, in order to either augment or
reduce the biological activity. The structure and topology of these
domains can all be used as a basis for the rational design of
pharmaceuticals (small molecule conventional drugs or novel
carbohydrate, lipid, DNA/RNA or protein-based high molecular weight
biological compounds) to modulate (increase or decrease) the
activity of PAMP and/or the PAMP PS1/PS2 complex, and/or the
activity of the PAMP/other protein complexes. For example, using
structural prediction calculations, possibly in conjunction with
spectroscopic data like nuclear magnetic resonance, circular
dichroism, and other physical-chemical structural data, or
crystallographic data, or both, one can generate molecular models
for the structure of PAMP. These models, in turn, are important for
rational drug design. Drug candidates generated using a rational
drug design program can then be applied for the treatment and/or
prevention of the above-mentioned diseases, and can be administered
through a variety of means including: as conventional small
molecules through enteral or parenteral routes; via inclusion in
liposome vehicles; through infusion in pumps inserted into various
organs (e.g., via Omaya pumps inserted into the cerebral
ventricles); via the transplantation of genetically-modified cells
expressing recombinant genes; or via the use of biological vectors
(e.g., retrovirus, adenovirus, adeno-associated virus, Lentivirus,
or herpes simplex virus-based vectors) which allow targeted
expression of appropriately modified gene products in selected cell
types. It should be noted that the recombinant proteins described
above may be the wild-type PAMP, a genetically-modified PAMP, a
wild-type PS1/PS2, a genetically-modified PS1/PS2, or a
specially-designed protein or peptide which is designed to interact
with either the functional domains of PAMP (or the
PAMP/PS1/PS2/other protein complex) or to interact with the domains
which modulate the activity of the functional domains of PAMP.
PAMP In Vitro and In Vivo Models
[0089] The PAMP nucleotide sequence can be used to make cell-free
systems, transfected cell lines, and animal models (invertebrate or
vertebrate) of the neurodegenerative and other diseases outlined
above. These animal and cell models may involve over-expression of
all or part of PAMP or PAMP mutants, e.g., as mini-gene cDNA
transgene constructs under the regulation of suitable promoter
elements carried in vectors such as cos-Tet for transgenic mice and
pcDNA (Invitrogen, California) in transfected cell lines. Animal
and cellular models can also be generated by via homologous
recombination mediated targeting of the endogenous gene to create
artificially mutant sequences (knock-in gene targeting); or loss of
function mutations (knock-out gene targeting); by translocation of
P-elements; and by chemical mutagenesis. Animal, cellular and
cell-free model systems can be used for a variety of purposes
including the discovery of diagnostics and therapeutics for this
disease.
[0090] Included within the scope of this invention is a mammal in
which two or more genes have been knocked out or knocked in, or
both. Such mammals can be generated by repeating the procedures set
forth herein for generating each knockout construct, or by breeding
to mammals, each with a single gene knocked out, to each other, and
screening for those with the double knockout genotype.
[0091] Regulated knockout animals can be prepared using various
systems, such as the tet-repressor system (see U.S. Pat. No.
5,654,168) or the Cre-Lox system (see U.S. Pat. No. 4,959,317 and
U.S. Pat. No. 5,801,030).
[0092] Transgenic mammals can be prepared for evaluating the
molecular mechanisms of PAMP, and particularly human PAMP function.
Such mammals provide excellent models for screening or testing drug
candidates. It is possible to evaluate compounds or diseases on
"knockout" animals, e.g., to identify a compound that can
compensate for a defect in PAMP activity. Alternatively, PAMP (or
mutant PAMP), alone or in combination with .beta.APP, PS1, PS2,
and/or Notch, or some other component (double or triple
transgenics) "knock-in" mammals can be prepared for evaluating the
molecular biology of this system in greater detail than is possible
with human subjects. Both technologies permit manipulation of
single units of genetic information in their natural position in a
cell genome and to examine the results of that manipulation in the
background of a terminally differentiated organism. These animals
can be evaluated for levels of mRNA or protein expression,
processing of .beta.APP, or development of a condition indicative
of inappropriate gene expression, e.g., Notch phenotype or another
phenotype as set forth above, or neurodegeneration, including
cognitive deficits, learning or memory deficits, or neuromuscular
deficits.
[0093] Various transgenic animal systems have been developed. Mice,
rats, hamsters, and other rodents are popular, particularly for
drug testing, because large numbers of transgenic animals can be
bred economically and rapidly. Larger animals, including sheep,
goats, pigs, and cows, have been made transgenic as well.
Transgenic D. melanogaster and C. elegans can also be made and,
using known genetic methods, may offer the ability to identify
upstream and downstream modifiers of a PAMP phenotype. Transgenic
animals can also be prepared by introducing the transgene on a
vector; such animals, which are not modified in the germ line and
are only transiently transgenic, naturally cannot pass along the
genetic information to their progeny.
[0094] In another series of embodiments, transgenic animals are
created in which (i) a human PAMP, or a mutant human PAMP, is
stably inserted into the genome of the transgenic animal; and/or
(ii) the endogenous PAMP genes are inactivated and replaced with
their human counterparts. See, e.g., Coffman, 1997; Esther et al.,
1996; Murakami et al., 1996. Such animals can be treated with
candidate compounds and monitored for the effects of such drugs on
PAMP cavity.
PAMP Gene Therapy
[0095] As discussed above, abnormalities in PAMP expression and/or
interactions with PS1/PS2/.beta.APP are associated with severe
neurological deficits. Thus, the present invention provides for
treatment of such deficits either with a drug discovered using a
screening assay or transgenic animal model, or both, as set forth
above, or by replacing a defective PAMP gene with a functional gene
by gene therapy.
[0096] A gene encoding PAMP, a PAMP mutant, or alternatively a
negative regulator of PAMP such as an antisense nucleic acid,
intracellular antibody (intrabody), or dominant negative PAMP
(which may be truncated), can be introduced in vivo, ex vivo, or in
vitro using a viral or a non-viral vector, e.g., as discussed
above. Expression in targeted tissues can be effected by targeting
the transgenic vector to specific cells, such as with a viral
vector or a receptor ligand, or by using a tissue-specific
promoter, or both. Targeted gene delivery is described in WO
95/28494, published October 1995.
[0097] Preferably, for in vivo administration, an appropriate
immunosuppressive treatment is employed in conjunction with the
viral vector, e.g., adenovirus vector, to avoid immuno-deactivation
of the viral vector and transfected cells. For example,
immunosuppressive cytokines, such as interleukin 12 (IL-12),
interferon-.gamma. (IFN.gamma.), or anti-CD4 antibody, can be
administered to block humoral or cellular immune responses to the
viral vectors (see, e.g., Wilson, 1995). In that regard, it is
advantageous to employ a viral vector that is engineered to express
a minimal number of antigens.
[0098] Herpes virus vectors. Because herpes virus is trophic for
cells of the nervous system (neural cells), it is an attractive
vector for delivery of function PAMP genes. Various defective
(non-replicating, and thus non-infectious) herpes virus vectors
have been described, such as a defective herpes virus 1 (HSV1)
vector (Kaplitt et al., 1991; International Patent Publication No.
WO 94/21807, published Sep. 29, 1994; International Patent
Publication No. WO 92/05263, published Apr. 2, 1994).
[0099] Adenovirus vectors. Adenoviruses are eukaryotic DNA viruses
that can be modified to efficiently deliver a nucleic acid of the
invention to a variety of cell types in vivo, and has been used
extensively in gene therapy protocols, including for targeting
genes to neural cells. Various serotypes of adenovirus exist. Of
these serotypes, preference is given to using type 2 or type 5
human adenoviruses (Ad 2 or Ad 5) or adenoviruses of animal origin
(see WO94/26914). Those adenoviruses of animal origin which can be
used within the scope of the present invention include adenoviruses
of canine, bovine, murine (example: Mav1, Beard et al., 1990),
ovine, porcine, avian, and simian (example: SAV) origin.
Preferably, the adenovirus of animal origin is a canine adenovirus,
more preferably a CAV2 adenovirus (e.g., Manhattan or A26/61 strain
(ATCC VR-800), for example). Various replication defective
adenovirus and minimum adenovirus vectors have been described for
gene therapy (WO94/26914, WO95/02697, WO94/28938, WO94/28152,
WO94/12649, WO95/02697 WO96/22378). The replication defective
recombinant adenoviruses according to the invention can be prepared
by any technique known to the person skilled in the art (Levrero et
al., 1991; EP 185 573; Graham, 1984; Graham et al., 1977).
Recombinant adenoviruses are recovered and purified using standard
molecular biological techniques, which are well known to one of
ordinary skill in the art.
[0100] Adeno-associated viruses. The adeno-associated viruses (AAV)
are DNA viruses of relatively small size which can integrate, in a
stable and site-specific manner, into the genome of the cells which
they infect. They are able to infect a wide spectrum of cells
without inducing any effects on cellular growth, morphology or
differentiation, and they do not appear to be involved in human
pathologies. The AAV genome has been cloned, sequenced and
characterized. The use of vectors derived from the AAVs for
transferring genes in vitro and in vivo has been described (see WO
91/18088; WO 93/09239; U.S. Pat. No. 4,797,368, U.S. Pat. No.
5,139,941, EP 488 528). The replication defective recombinant AAVs
according to the invention can be prepared by co-transfecting a
plasmid containing the nucleic acid sequence of interest flanked by
two AAV inverted terminal repeat (ITR) regions, and a plasmid
carrying the AAV encapsidation genes (rep and cap genes), into a
cell line which is infected with a human helper virus (for example
an adenovirus). The AAV recombinants which are produced are then
purified by standard techniques.
[0101] Retrovirus vectors. In another embodiment the gene can be
introduced in a retroviral vector, e.g., as described in Anderson
et al., U.S. Pat. No. 5,399,346; Mann et al., 1983; Temin et al.,
U.S. Pat. No. 4,650,764; Temin et al., U.S. Pat. No. 4,980,289;
Markowitz et al., 1988; Temin et al., U.S. Pat. No. 5,124,263; EP
453242, EP178220; Bernstein et al., 1985; McCormick, 1985;
International Patent Publication No. WO 95/07358, published Mar.
16, 1995, by Dougherty et al.; and Kuo et al., 1993. The
retroviruses are integrating viruses which infect dividing cells.
The retrovirus genome includes two LTRs, an encapsidation sequence
and three coding regions (gag, pol and env). In recombinant
retroviral vectors, the gag, pol and env genes are generally
deleted, in whole or in part, and replaced with a heterologous
nucleic acid sequence of interest. These vectors can be constructed
from different types of retrovirus, such as MoMuLV ("murine Moloney
leukemia virus"), MEV ("murine Moloney sarcoma virus"), HaSV
("Harvey sarcoma virus"); SNV ("spleen necrosis virus"); RSV ("Rous
sarcoma virus") and Friend virus. Suitable packaging cell lines
have been described in the prior art, in particular the cell line
PA317 (U.S. Pat. No. 4,861,719); the PsiCRIP cell line (WO
90/02806) and the GP+envAm-12 cell line (WO 89/07150). In addition,
the recombinant retroviral vectors can contain modifications within
the LTRs for suppressing transcriptional activity as well as
extensive encapsidation sequences which may include a part of the
gag gene (Bender et al., 1987). Recombinant retroviral vectors are
purified by standard techniques known to those having ordinary
skill in the art.
[0102] Retrovirus vectors can also be introduced by recombinant DNA
viruses, which permits one cycle of retroviral replication and
amplifies transfection efficiency (see WO 95/22617, WO 95/26411, WO
96/39036, WO 97/19182).
[0103] Lentivirus vectors. In another embodiment, lentiviral
vectors are can be used as agents for the direct delivery and
sustained expression of a transgene in several tissue types,
including brain, retina, muscle, liver and blood. The vectors can
efficiently transduce dividing and non-dividing cells in these
tissues, and maintain long-term expression of the gene of interest.
For a review, see, Naldini, 1998; see also Zufferey, et al., 1998).
Lentiviral packaging cell lines are available and known generally
in the art. They facilitate the production of high-titer lentivirus
vectors for gene therapy. An example is a tetracycline-inducible
VSV-G pseudotyped lentivirus packaging cell line which can generate
virus particles at titers greater than 106 IU/ml for at least 3 to
4 days (Kafri, et al., 1999). The vector produced by the inducible
cell line can be concentrated as needed for efficiently transducing
nondividing cells in vitro and in vivo.
[0104] Non-viral vectors. A vector can be introduced in vivo in a
non-viral vector, e.g., by lipofection, with other transfection
facilitating agents (peptides, polymers, etc.), or as naked DNA.
Synthetic cationic lipids can be used to prepare liposomes for in
vivo transfection, with targeting in some instances (Felgner, et.
al., 1987; Felgner and Ringold, 1989; see Mackey, et al., 1988;
Ulmer et al., 1993). Useful lipid compounds and compositions for
transfer of nucleic acids are described in International Patent
Publications WO95/18863 and WO96/17823, and in U.S. Pat. No.
5,459,127. Other molecules are also useful for facilitating
transfection of a nucleic acid in vivo, such as a cationic
oligopeptide (e.g., International Patent Publication WO95/21931),
peptides derived from DNA binding proteins (e.g., International
Patent Publication WO96/25508), or a cationic polymer (e.g.,
International Patent Publication WO95/21931). Recently, a
relatively low voltage, high efficiency in vivo DNA transfer
technique, termed electrotransfer, has been described (Mir et al.,
1998; WO 99/01157; WO 99/01158; WO 99/01175). DNA vectors for gene
therapy can be introduced into the desired host cells by methods
known in the art, e.g., electroporation, microinjection, cell
fusion, DEAE dextran, calcium phosphate precipitation, use of a
gene gun (ballistic transfection), or use of a DNA vector
transporter (see, e.g., Wu et al., 1992; Wu and Wu, 1988; Hartmut
et al., Canadian Patent Application No.2,012,311, filed Mar. 15,
1990; Williams et al., 1991). Receptor-mediated DNA delivery
approaches can also be used (Curiel et al., 1992; Wu and Wu, 1987).
U.S. Pat. Nos. 5,580,859 and 5,589,466 disclose delivery of
exogenous DNA sequences, free of transfection facilitating agents,
in a mammal.
EXAMPLES
[0105] The present invention will be further understood by
reference to the following examples, which are provided as
exemplary of the invention and not by way of limitation.
Example 1
A Novel PAMP that Mediates .beta.APP Processing and Notch/Glp1
Signal Transduction
[0106] This example shows that both PS1 and PS2 interact with a
novel Type I transmembrane protein, PAMP, and that this novel
protein also interacts with .alpha.- and .beta.-secretase derived
fragments of .beta.APP. We also show that abolition of functional
expression of the C. elegans homologue of the protein leads to a
developmental phenotype (anterior pharynx aph-2) which is thought
to be due to inhibition of the glp/Notch signaling pathway. This
novel protein is therefore positioned to mediate both the gain of
function and loss of function phenotypes associated with presenilin
missense mutations and presenilin knockouts, respectively.
Materials and Methods
[0107] Antibodies against PS1, PS2 and .beta.APP. An antibody,
termed 1142, directed against PS1, was raised to a peptide segment
corresponding to residues 30-45 of PS1 (Levesque et al., 1998; Yu
et al., 1998). The peptide was synthesized by solid-phase
techniques and purified by reverse phase high pressure liquid
chromatography (HPLC). Peptide antigens were linked to keyhole
limpet hemocyanin (KLH) and used, in combination with complete
Freud's adjuvant, to innoculate New Zealand White rabbits. Antisera
from three rabbits was pooled, ammonium precipitated and the
antibody was purified with Sulfo-link (Pierce) agarose-peptide
affinity columns. Other antibodies used include antibody 369, a
polyclonal rabbit-anti-human antibody directed against the
C-terminus of human .beta.APP (Buxbaum et al., 1990); antibody 14
(Ab14), a rabbit polyclonal antibody raised against residues 1-25
of human PS1 (Seeger et al., 1997); antibody .alpha.-PS1-CTF, a
polyclonal rabbit antibody directed against the PS1 loop; and
antibody DT2, a monoclonal antibody raised to a GST-fusion protein
containing the PS2 N-terminal sequence from residues 1-87.
[0108] Preparation of presenilin associated components. To identify
membrane associated components of the presenilin complex, an
immunoaffinity procedure was used to extract PS1 and tightly
associated membrane proteins from semi-purified intracellular
membrane fractions. Human embryonic kidney cells (HEK) 293 (ATCC)
with a stable over-expression of moderate level wild type human
PS1, were grown to confluence, washed twice with ice-cold
phosphate-buffered saline, and then homogenized with Buffer A (0.25
M sucrose, 20 mM HEPES pH 7.2, 2 mM EGTA, 2 mM EDTA, 1 mM DTT, and
a protease inhibitor cocktail containing 5 mg/ml each of Leupeptin,
Antipain, pepstatin A, Chymostatin, E64, Aprotinin, and 60 mg/ml
4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF)). The cell
homogenates were centrifuged 1000.times.g for 10 minutes to remove
cell debris. The supernatant was then centrifuged 10,000.times.g
for 60 minutes. The resulting membrane pellet was resuspended in
Buffer B (20 mM HEPES pH 7.2, 1 M KCl, 2 mM EGTA, 2 mM EDTA, 1 mM
DTT, and protease inhibitor cocktail as above) and incubated for 45
minutes with agitation at 4.degree. C. Cell membranes were
collected again by centrifugation at 107,000.times.g for 60
minutes. The cell membranes were then lysed on ice for 60 minutes
with Buffer C (1% Digitonin, 20 mM HEPES pH 7.2, 100 mM KCl, 2 mM
EGTA, 2 mM EDTA, 1 mM DTT, and protease inhibitors cocktail). After
spinning 10,000.times.g for 15 minutes, the protein extract was
adjusted with Buffer C to contain 5 mg/ml protein. A total of 0.5 g
of protein was obtained.
[0109] Isolation. The extracted proteins were then subjected to
fractionation with 10-40% glycerol gradient containing 0.5%
Digitonin as described (Yu et al., 1998). After being verified by
Western blotting with anti-PS1 antibodies, the peak fractions
containing PS1 were pooled and incubated overnight with Protein A/G
agarose coupled with either antibody 1142 or a control IgG purified
from preimmune rabbit serum. The Protein A/G agarose beads were
washed three times with Buffer D (1% Digitonin, 20 mM HEPES pH 7.2,
100 mM KCl, protease inhibitors cocktail), and three times with
Buffer E (0.5% Digitonin, 0.5% CHAPS, 20 mM HEPES pH 7.2, 100 mM
KCl, 10 mM CaCl.sub.2, 5 mM MgCl.sub.2, and the protease inhibitor
cocktail as above). Isolated protein complexes were eluted from the
beads with 0.1M Glycine-HCl, pH 3.0, and then neutralized with 1M
Tris. Proteins were then separated by Tris-Glycine SDS-PAGE gels
and stained with silver stain and Coomassie Blue stain. The
staining of the immuno-purified proteins displayed two intense
bands in addition to those of the presenilin holoprotein and its
fragments.
[0110] Sequence analysis. Individual protein bands were cut out and
analyzed with solid-phase extraction capillary electrophoresis mass
spectrometry/mass spectrometry (SPE-CE-MS/MS). Briefly, protein
bands were first digested in-gel with trypsin; the digested
proteins were extracted and dried in a speed vacuum down to
concentrate the peptides; and the peptides thereafter separated
with micro LC and analyzed by on-line tandem mass spectrometry
(Figeys et al., 1999). Nucleotide and amino acid sequence homology
searches were conducted using the BLAST algorithm, and motif
analyses performed using the program BLOCKS.
[0111] General transfection and analysis methods. Based on the
human PAMP sequence, public databases (e.g., GenBank; see
ncbi.nlm.nih.gov on the World-Wide Web (www)) were searched for
homologous ESTs (SEQ ID NOs:3-10), which were collected into a few
contigs. These contigs all turned out to be correct, but did not
cover full-length mouse and D. melanogaster cDNAs.
[0112] Full length murine (SEQ ID NO: 15), human (SEQ ID NO: 13)
and D. melanogaster (SEQ ID NO: 17) PAMP cDNAs were obtained using
oligonucleotides designed from partial cDNA/EST sequences in public
databases to screen appropriate cDNA libraries, for 5'RACE, and/or
for RT-PCR experiments. A PAMP expression construct was generated
by inserting human PAMP cDNA in-frame with the V5 epitope of pcDNA6
(Invitrogen) at the C-terminus of PAMP. HEK293 cells with a stable
expression of PS1/PS2 and .beta.APP.sub.sw were transiently
transfected with either V5-tagged PAMP or empty plasmid (mock
transfection control). Duplicate experiments were performed by: (1)
transient transfection of V5-PAMP and .beta.APP.sub.695 (or empty
vector plus .beta.APP.sub.695 as a mock transfection control) into
murine embryonic fibroblasts stably infected with human PS1
expressed from a retroviral vector construct (Clontech, CA); or (2)
transient transfection of V5-PAMP (or an empty plasmid) into HEK293
cell lines with a stable expression of the C-terminal 99 amino
acids of .beta.APP with a preceding artificial signal peptide
(spC100-APP) together with either wild type PS1, PS1-L392V, or
PS1-D385A. Cells were lysed with a Digitonin lysis buffer or with
1% NP40, and the protein extracts were subjected to gradient
fraction, immunoprecipitation or direct Western blotting as
described (Yu et al., 1998). PS1 was immunodetected or
immunoprecipitated with antibodies 14 or a-PS1-CTF; and PS2 was
immunodetected or immunoprecipitated with antibody DT2.
FL-.beta.APP and its C-terminal .alpha.- and .beta.-secretase
derivatives were detected using antibody 369.
Results
[0113] Isolation of PAMP. Immunoprecipitation of PS1 protein
complexes, followed by SDS-PAGE with Coomassie Blue and silver
staining, yielded two intense bands in addition to presenilin
holoprotein. These bands were characterized by mass spectroscopy
analysis. Mass spectroscopy analysis revealed several armadillo
repeat containing peptides, (previously known to functionally
interact with presenilins (Yu et al., 1998; Zhou et al. (1), 1997;
Nishimura M, et al., 1999), and a novel peptide (PAMP) which had a
sequence identified to that predicted for an anonymous, partial
cDNA (Genbank; Accession No. D87442). The cDNA sequence predicted
an open reading frame of 709 amino acids (SEQ ID NO: 14), which
contains a putative N-terminal signal peptide, a long N-terminal
hydrophilic domain with sequence motifs for glycosylation,
N-myristoylation and phosphorylation, a .about.20 residue
hydrophobic putative transmembrane domain, and a short hydrophilic
C-terminus of 20 residues (FIGS. 1A and 1B).
[0114] Orthologous PAMP proteins. The PAMP amino acid sequence had
no significant homology to other proteins within available
databases, except for a hypothetical C. elegans protein
(www.ncbi.nih.gov; Accession No. Q23316) (p=2.times.10.sup.-28;
identity=22%; similarity=39%) (SEQ ID NO: 12) ascertained from a
genomic DNA sequence (FIGS. 1A and 1B). In addition to strong
primary amino acid sequence conservation, this C. elegans protein
has a very similar topology to human PAMP, suggesting that it is
the nematode orthologue of human PAMP.
[0115] In the absence of functional clues arising from homologies
to other known proteins, the predicted amino acid sequences of the
murine (SEQ ID NO: 16) and D. melanogaster (SEQ ID NO: 18)
orthologues of PAMP were cloned and examined. The four orthologous
PAMP proteins had a similar topology and significant sequence
conservation near residues 306-360, 419-458, and 625-662 of human
PAMP (SEQ ID NO: 14) (FIGS. 1A and 1B). Motif analysis of these
conserved domains revealed a weak similarity (strength=1046)
between residues 625-641 (ARLARALSPAFELSQWS; SEQ ID NO: 19) of
mouse and human PAMP to cyclic nucleotide binding domains. While
the putative transmembrane domain sequences were not highly
conserved, all four orthologues contained a conserved serine
residue within this hydrophobic domain. Finally, there were four
conserved cysteine residues in the--terminal hydrophilic domain
(Cys.sub.195, Cys.sub.213, Cys.sub.230, and Cys.sub.248 in human
PAMP) which had a periodicity of 16-17 residues in the N-terminus,
and may form a functional domain (e.g., a metal binding domain or
disulfide bridges for stabilizing the tertiary structure of
PAMP/PAMP complexes).
[0116] Interaction of PAMP with presenilin 1. To confirm the
authenticity of the PAMP:PS1 interaction, HEK293 cells were
transiently transfected with PAMP cDNA (SEQ ID NO: 13) tagged at
the 3'-end with a V5-epitope encoded from the pcDNA6 vector. The
conditioned media were collected 20 hr after transient transfection
with PAMP (or with empty vector), and the A.beta..sub.40 and
A.beta..sub.42 levels were measured by ELISA (Zhang L, et al.,
1999). In Western blots of lysates of these cells, the use of
anti-V5 (Invitrogen, CA) and enhanced chemiluminescence (Amersham)
detected a V5-immunoreactive band of .about.110 kDa which was
reduced to .about.80 kDa following Endo H digestion (equivalent to
the size predicted from the PAMP amino acid sequence), confirming
the predicted glycosylation of PAMP. In addition, a series of about
7-10 kDa fragments were observed, which are predicted to contain
the TM domain and short C-terminus of PAMP plus the 3 kDa
V5-epitope. These C-terminal derivatives may be authentic cleavage
products of full-length PAMP, or, alternatively, a proteolytic
artifact arising from the attachment of the V5-epitope to the
C-terminus of PAMP.
[0117] Reciprocal immunoprecipitation studies in cells expressing
combinations of transfected V5-tagged-PAMP, transfected wild type
or mutant PS1, transfected wild type PS2, or endogenous
presenilins, confirmed the PS1:PAMP interaction, and showed a
similar interaction between PAMP and PS2. In contrast,
immunoprecipitation of other ER-resident proteins (e.g., calnexin)
failed to show any evidence of an interaction between these
proteins and PAMP. Glycerol velocity gradient analysis of the
native conformation of PAMP revealed that PAMP was co-eluted into
the same high molecular weight fractions as PS1 and PS2, indicating
that it is an authentic component of the high molecular weight
presenilin protein complexes. These biochemical data were supported
by immunocytochemical studies, which showed that transfected PAMP
and endogenous PS1 strongly co-localized in the ER and Golgi in
MDCK canine kidney/epithelial cells (ATCC). Similar studies with
PS2 confirmed that PAMP also tightly associates with both
endogenous PS2 in human brain and with transfected PS2 in HEK293
cells.
[0118] The PAMP gene. Chromosomal locations and genetic map
positions of the murine and human PAMPS were obtained from public
genetic and transcriptional maps (www.ncbi.nlm.nih.gov). The gene
encoding PAMP is located on human chromosome 1 near the genetic
markers D1S1595 and D1S2844. The 5'-end of the PAMP gene is
embedded in the 5'-end of the coatmer gene encoded on the opposite
strand. The human PAMP gene is close to a cluster of markers which
have yielded positive, but sub-significant evidence for linkage to
or association with Alzheimer Disease in two independent genome
wide surveys (Kehoe et al., 1999). The murine PAMP maps within a
700 Kb interval of murine chromosome 1 which contains the gene
defect associated with Looptail phenotype in mice (Underhill et
al., 1999). Mice heterozygous for Looptail show developmental
defects in dorsal axial structures including notochord, brain,
spinal cord, and somites (Greene et al., 1998), which are
reminiscent of those observed in PS1.sup.-/- mice (Shen J, et al.,
1997; Wong et al., 1997). These observations suggest that the
presenilin: PAMP complex may be involved in both .beta.APP and
Notch processing.
[0119] C. elegans homolog of PAMP. The C. elegans homolog of PAMP
corresponds to the aph-2 gene. Mutations in aph-2 have been
identified in a screen for mutants with phenotypes identical to
embryonic mutant phenotypes caused by loss of glp-1 activity, i.e.,
lack of an anterior pharynx, e.g. cDNA clone. The EST corresponding
to aph-2, (cDNA clone yk477b8, kindly provided by Y. Kohara,
National Institute of Genetics, Japan) was sequenced and the coding
region (SEQ ID NO: 11) found to match exactly the Genefinder
prediction made by the C. elegans sequencing consortium (Genbank;
Accession No. Z75714). Double stranded RNA interference (RNAi)
confirmed the mutant phenotype of aph-2. Sense and antisense RNA
were transcribed in vitro from PCR product amplified from the phage
yk477b8. After annealing equal quantities of sense and antisense
products, the dsRNA product made was injected into L4 stage
wild-type worms. The chosen line of worms, designated lin-12(n302)
(Greenwald and Seydoux, 1990; Greenwald, et al., 1983) was obtained
from the Caenorhabditis Genetics Center. Injected animals were
transferred to fresh plates daily and the progeny scored at least
36 hours after injection for the embryonic lethal phenotype and 4-5
days after injection for the egg-laying phenotypes. Animals
injected with dsRNA from yk477b8 template produced eggs that lacked
an anterior pharynx. These results support the notion that
aph-2/PAMP contributes to cell interactions mediated by glp-1/Notch
in the embryo.
[0120] Functional role for the PAMP:presenilin complexes in
.beta.APP processing. To examine a functional role for the
PAMP:presenilin complexes in .beta.APP processing, the interactions
between PAMP, PS1, and .beta.APP, and its derivatives were
investigated. The cell lines used were transiently transfected with
V5-tagged PAMP, and stably expressing wild type .beta.APP.sub.695
in addition to wild type PS1, wild type PS2, PS1-L392V mutant, or
PS1-D385A mutant. The PS1-L392V mutation is a pathogenic mutation
associated with familial AD (Sherrington et al., 1995) and with
increased secretion of A.beta..sub.42 (Scheuner et al., 1996;
Citron et al., 1997). The PS1-D385A mutation is a loss of function
mutation associated with inhibition of PS1 endoproteolysis and a
decrease in .gamma.-secretase activity (Wolfe et al., 1999). The
conditioned media were collected 20 hr after transient transfection
with PAMP (or with empty vector), and the A.beta..sub.40 and
A.beta..sub.42 levels were measured by ELISA (Zhang et al., 1999).
Analysis of Western blots from these co-immunoprecipitation
experiments revealed that PAMP holoprotein (and C-terminally tagged
proteolytic fragments of PAMP) interacted in equivalent degrees
with wild type PS1, wild type PS2, PS1-L392V mutant, and PS1-D385A
mutant proteins. In addition, PAMP holoprotein and the C-terminal
proteolytic fragments of PAMP also co-immunoprecipitated with the
C-terminal proteolytic fragments of .beta.APP but not .beta.APP
holoprotein in lysates of cells expressing either .beta.APP
holoprotein or just the C-terminal 99 amino acids of .beta.APP.
Significantly, compared to cells expressing equivalent quantities
of wild type PS1, cell lines expressing pathogenic mutations of PS1
showed increased amounts of C-terminal .beta.APP fragments
co-immunoprecipitating with PAMP. Conversely, cell lines expressing
the loss-of-function PS1-D385A mutation showed greatly reduced
amounts of C-terminal .beta.APP derivatives co-immunoprecipitating
with PAMP despite the presence of very large amounts of C-terminal
.beta.APP derivatives in these cells.
[0121] These results were confirmed in HEK293 cells over-expressing
either .beta.APP.sub.Swedish or the SpC99-.beta.APP cDNA. The
latter encodes the C-terminal 99 residues of .beta.APP
(corresponding to the products of .beta.-secretase cleavage) plus
the .beta.APP signal peptide. The interaction of PAMP appears much
stronger with C99-.beta.APP than that with C83-.beta.APP. However,
C83-.beta.APP is much less abundant in these cells (FIG. 6b, middle
panel, lanes 1-4). In fact, PAMP does interact with both C99- and
C83-.beta.APP stubs (see FIG. 6c, lane 9 and FIG. 8d).
Cumulatively, these results indicate that PAMP likely interacts
with the C-terminal derivatives of .beta.APP which are the
immediate precursors of A.beta. and p3. However, of greater
interest, the genotype of the co-expressed PS1 molecule dynamically
influenced the interaction between PAMP and C99-/C83-.beta.APP
stubs. Thus, more C-terminal .beta.APP fragments
co-immunoprecipitated with PAMP in cells expressing the
FAD-associated PS1-L392V mutation compared to cells expressing wild
type PS1 (and equivalent quantities of nicastrin and
C99-.beta.APP). Conversely, much less C-terminal .beta.APP
derivatives co-immunoprecipitated with PAMP in cell lines
expressing the loss-of-function PS1-D385A mutation (despite the
presence of very large amounts of C-terminal .beta.APP derivatives
in these cells). These effects are more easily seen in cells
over-expressing the C99-.beta.APP construct. However, similar but
less pronounced differences were also observed in cells
over-expressing full-length .beta.APP.sub.Swedish. More
importantly, the PS1-sequence-related differences in the
interaction of PAMP with C-terminal .beta.APP derivatives were most
evident within 24 hours of transient transfection of PAMP. By 72
hours, the PS1-sequence-related differences were largely abolished.
This dynamic change in the interaction of PAMP with
C99/C83-.beta.APP was not due to changes in the levels of PS1,
C-terminal .beta.APP derivatives or PAMP. One interpretation of
these results is that the presenilins may be dynamically involved
in regulating or loading PAMP with the substrates of
.gamma.-secretase.
[0122] Presenilin binding domains of PAMP. In transiently
transfected cells (in which the 7-10 kDa C-terminal of PAMP can be
detected), anti-PS1 immunoprecipitation products contain both full
length PAMP and the .about.7-10 kDa C-terminal PAMP fragments.
Similarly, in these cells, immunoprecipitation with antibodies to
the C-terminus of .beta.APP (Ab369) also renders C-terminal
nicastrin epitopes. The TM domain of PAMP is not highly conserved
in evolution. These results suggest that the C99-/C83-.beta.APP and
PS1/PS2-binding domain(s) of PAMP are in the TM domain or
C-terminus.
Discussion
[0123] The above results indicate that PAMP is a component of the
PS1 and PS2 intracellular complexes. The observations that PAMP
also binds to the C-terminal fragment of .beta.APP (arising from
.alpha.- and .beta.-secretase cleavage of full length .beta.APP),
that the degree of binding of these fragments to PAMP is modulated
by mutations in PS1, and that the direction of this modulation is
congruent with the effects of each mutant of A.beta. production
(i.e., the pathogenic L392V mutation increases binding to PAMP and
increases A.beta..sub.42 production whereas the D385A mutation has
the opposite effects) strongly argues that PAMP is part of a
functional complex involved in processing of C-terminal .beta.APP
derivatives. Similarly, the observation that inhibition of PAMP
expression in C. elegans leads to a phenotype similar to that of
glp/Notch loss of function, argues that PAMP, like PS1 and PS2, is
also a functional component of the pathways involved in processing
of Notch. This conclusion is strengthened by the fact that the
murine PAMP gene maps within a 700 kb interval on murine chromosome
1 which carries the Looptail mutant, and is thus likely to be the
site of the Looptail mutation. Looptail has a number of phenotypic
similarities to those of Notch and PS1 knockouts in mice. Because
Looptail is a model of human spinal cord malformations including
spina bifida, PAMP biology may also provide some useful insights
into this neurological developmental defect as well.
[0124] At the current time the exact role of PAMP in the
presenilin-complex-mediated processing of .beta.APP and Notch-like
molecules is not fully defined. Inspection of the primary amino
acid sequence of PAMP does not reveal very strong homologies to
known proteases. However, the recombinant expression systems of the
invention permit evaluation of three-dimensional structure of PAMP;
it is possible that PAMP itself has a protease activity. However,
it is currently more plausible that PAMP plays another role in
.beta.APP and Notch processing. Thus, PAMP may be involved in the
function of PS1 and PS2 complexes by binding substrates for
.gamma.-secretase. The efficacy of this binding is clearly
modulated by PS1 mutations in a direction which is commensurate
with the effect of these mutations on .gamma.-secretase activity.
Alternatively, PAMP may have a regulatory role on the activity of
the presenilin complexes. This is consistent with the observation
that residues 625-641 of human and murine PAMP contain a motif
similar to cyclic nucleotide binding domains of several other
unrelated proteins.
[0125] Regardless of its precise role, it is clear that PAMP and
PS1 both play important roles in .gamma.-secretase mediated
processing of .beta.APP. Hence, knowledge of PAMP and its biology
will now serve as a target for efforts to manipulate the function
of the presenilin complexes in patients with schizophrenia and/or
Alzheimer Disease and related disorders, patients with malignancies
(in which the presenilins have been implicated by virtue of a role
in programmed cell death), and in disorders of development
especially of the spinal cord and brain (in view of the known
effects of PS1 knockout and the strong likelihood that PAMP is the
site of Looptail mutations in mice). In particular, knowledge of
the domains of PAMP involved in binding presenilins and .beta.APP
derivatives (which currently appears to be located within the
C-terminal transmembrane and hydrophilic domains of PAMP) and the
identification of putative ligands interacting with the conserved
domains at the hydrophilic N-terminus of PAMP will considerably
expedite this goal.
[0126] We have found that the strength of the interaction between
PAMP and the C-terminal fragments of .beta.APP (which is the
precursor A.beta.) is determined by the genotype at PS1. Thus,
clinical mutations in PS1 cause Alzheimer Disease and an increase
in the production of A.beta..sub.42 are associated with increased
binding of the C-terminal fragments of .beta.APP to PAMP.
Conversely, loss of function mutations in PS1 (Asp385Ala) which
inhibit .gamma.-secretase cleavage of C-terminal fragments of
.beta.APP, are associated with abolition of the interaction between
PAMP and the C-terminal fragments of .beta.APP.
[0127] Finally, the apparent C-terminal proteolytic derivatives of
PAMP could either be authentic, or simply artefacts due to the V-5
tag. If they are authentic, this observation raises the possibility
that PAMP may undergo post-translational processing events which
are potentially similar to those of .beta.APP and/or Notch. Three
observations support our discovery of PAMP. First, in contrast to
.beta.APP and Notch, which are not major constituents of the high
molecular weight presenilin complexes, and which can only be
inconsistently shown to be directly associated with PS1/PS2, PAMP
is a major stoichiometric component of the presenilin complexes.
Second, PAMP selectively interacts only with C-terminal derivatives
of .beta.APP which are substrates for .gamma.-secretase cleavage,
and this interaction is modulated by PS1 mutations in a way which
reflects the functional consequences of these PS1 mutations. Third,
inhibition of PAMP expression in C. elegans leads to a disease
phenotype likely to be in the glp/Notch signaling pathway.
Example 2
PAMP Mutants
[0128] Site-directed mutagenesis was used to generate the following
artificial mutations in PAMP:
[0129] Cys: PAMP.sub.C230A in the 4 conserved cystine motif
[0130] DYIGS: PAMP.sub.D336A/Y337A in the central conserved
region
[0131] D369L: PAMP.sub..DELTA.312-369 in the central conserved
region
[0132] D340X: PAMP.sub..DELTA.312-340 in the central conserved
region
[0133] YDT: PAMP.sub.D458A in the putative `aspartyl protease` DTA
site
[0134] SPAF: PAMP.sub.P633A/F635A in the SPAF motif
[0135] TM: PAMP.sub.S683A in the TM domain
[0136] C3D: PAMP.sub..DELTA.630-668 in the conserved region
adjacent to the TM domain
[0137] To further examine the role of PAMP in .beta.APP processing,
we inserted PAMP cDNAs, harboring the above mutations as well as
normal/wild type PAMP (PAMP.sub.wt) cDNA and the cDNA for an
unrelated protein (LacZ), in frame into pcDNA6 vectors. A series of
HEK293 cell lines stably expressing endogenous PS1,
.beta.APP.sub.Swedish and either wild type PAMP or PAMP constructs
in which various conserved domains had been mutated or deleted,
were then created by transfection. PAMP expressing cells were
selected with lasticidin to generate stable cell lines. Conditioned
media from these cell lines were collected after 6-24 hours and
A.beta..sub.40 and A.beta..sub.42 were measured by ELISA.
[0138] In the PAMP.sub.D336A-Y337A mutant, both A.beta..sub.40 and
A.beta..sub.42 levels were increased, and there was also a 68%
increase in A.beta..sub.42/A.beta..sub.40 ratio which is very
similar to that observed in clinical mutations in APP, PS1, and
PS2, associated with early onset Alzheimer Disease. The
A.beta..sub.42/A.beta..sub.40 ratio was also increased in one cell
line expressing the PAMP.sub.C230A mutant.
[0139] In contrast, both the total A.beta..sub.42 and
A.beta..sub.40 levels and the A.beta..sub.42/A.beta..sub.40 ratio
were massively reduced (to only 18% of the control) in the
PAMP.sub..DELTA.312-369 mutant. A similar but less profound
reduction of both the total A.beta..sub.42 and A.beta..sub.40
levels and the A.beta..sub.42/A.beta..sub.40 ratio was observed in
the conditioned medium from the PAMP.sub..DELTA.312-340 cell
lines.
[0140] There is no apparent difference in A.beta..sub.42 or
A.beta..sub.40 levels, or in the A.beta..sub.42/A.beta..sub.40
ratio, when the PAMP.sub.wt, PAMP.sub.D458A,
PAMP.sub..DELTA.630-668, PAMP.sub.P633A/F635A, and PAMP.sub.S683A
cells were compared to control lines (expressing LacZ, or empty
vector).
[0141] Thus, certain PAMP mutants cause biochemical changes similar
to those induced by mutations in the .beta.APP, PS1, and PS2 genes
which give rise to AD, and which may be implicated also in
schizophrenia. These artificial PAMP mutations can therefore be
used to generate cellular and other model systems to design
treatments and preventions for schizophrenia, AD and other
neurodegenerative and/or neuropsychiatric disorders. These
mutations also show that PAMP is involved in the pathogenesis of
AD, and may provide information for the design of specific
molecular diagnostics or therapeutics for schizophrenia, AD, and
other neurological disorders.
[0142] When compared to mock-transfected or LacZ transfected cells,
overexpression of wild type PAMP, and overexpression of most PAMP
mutation- or deletion-constructs had no significant effect on
A.beta. secretion. However, missense mutation of the conserved
DYIGS motif to AAIGS (residues 336-340 of human PAMP) caused a
significant increase in A.beta..sub.42 secretion, a smaller
increase in A.beta..sub.40 secretion, and an increase in the
A.beta..sub.42/A.beta..sub.40 ratio (p<0.001; Table 2). This
increase in A.beta..sub.42 production was equivalent to that of
FAD-related missense mutations in PS1. Conversely, deletion of the
DYIGS domain in two independent constructs (PAMP.sub..DELTA.312-369
and PAMP.sub..DELTA.312-340) caused a significant reduction in both
A.beta..sub.42 and A.beta..sub.40 secretion which was more profound
in PAMP.sub..DELTA.312-3369 cells than in PAMP.sub..DELTA.312-340
cells (Table 2). The magnitude of the reduction in A.beta.
secretion in PAMP.sub..DELTA.312-369 cells was equivalent to that
observed with the PS1-D385A loss-of-function mutation. Somewhat
unexpectedly, and in contrast to PS1.sup.-/- and PS1-D385A cells,
the reduction in A.beta. secretion in NCT.sub..DELTA.312-369 and
NCT.sub..DELTA.312-340 cells was not accompanied by the expected
accumulation of C99- and C83-.beta.APP stubs. Since there was no
consistent change in the levels of soluble .beta.APP
(.beta.APP.sub.s) in the conditioned medium of any of the PAMP
mutant cells, the most probable explanation for this result is that
C99- and C83-.beta.APP stubs which do not enter the PAMP:presenilin
complex for .gamma.-secretase cleavage to A.beta. may be degraded
by other pathways.
[0143] The effects of PAMP mutations on A.beta. secretion were not
due to trivial explanations such as differences in the levels of
PAMP, .beta.APP holoprotein, or PS1/PS2. None of these mutations
caused any consistent, detectable change in the amount of APP.sub.s
in conditioned medium or in the amount of C99/C83.beta.APP that
could be co-immunoprecipitated with PAMP. However, both the
PAMP.sub..DELTA.312-369 mutant and the PAMP.sub..DELTA.312-340
deletion mutant significantly reduced the amount of PS1 which could
be co-immunoprecipitated with PAMP. Interestingly, the reduction in
efficiency of binding to PS1 was proportional to the reduction in
A.beta. secretion induced by each deletion mutant. Multiple
mechanisms underlying the effect of mutations in the first
conserved domain can explain these results. This domain contains no
obvious functional motifs (e.g., for glycosylation etc.), nor does
it have significant sequence homology to other known proteins.
Consequently, the three functionally active PAMP mutations either
affect a presenilin-binding domain in PAMP, or affect a specific
regulatory domain of PAMP which modulates both direct binding of
PAMP to PS1 and the subsequent .gamma.-secretase-mediated cleavage
of PAMP-bound C99- and C83-.beta.APP stubs. TABLE-US-00002 TABLE 2
A.beta..sub.42/A.beta..sub.40 Transfection Normalized
A.beta..sub.42 Normalized A.beta..sub.40 Ratio Mock (LacZ/empty 1.0
1.0 1.0 vector) wild type PAMP 1.03 .+-. 0.09 1.05 .+-. 0.07 0.99
.+-. 0.07 D336A/Y337A 3.09 .+-. 0.59 1.61 .+-. 0.19 1.81 .+-. 0.15
(p < 0.001) (p = 0.001) (p < 0.001) PAMP.sub..DELTA.312-369
0.05 .+-. 0.04 0.31 .+-. 0.06 0.09 .+-. 0.05 (p < 0.001) (p <
0.001) (p < 0.001) PAMP.sub..DELTA.312-340 0.33 .+-. 0.04 0.55
.+-. 0.04 0.59 .+-. 0.06 (p = 0.002) (p = 0.001) (p = 0.003)
Example 3
PAMP Interaction with Notch
[0144] PAMP interaction with Notch was studied using a
Notch-cleavage assay (De Strooper, 1999). Notch cDNA was tagged
with myc to the membrane-portion of Notch or to the soluble
proteolytic derivative called Notch intra-cellular domain (NICD).
V5-epitope-tagged PAMP and myc-tagged-Notch cDNAs were
co-transfected into HEK293 cells. Thereafter, V5-tagged-PAMP was
immunoprecipitated with anti-V5-antibodies, and the
immunoprecipitation products investigated for myc-tagged proteins.
In the immunoprecipitate, myc-tagged-Notch was found, but not
myc-tagged-NICD. This result indicates a specific interaction
between PAMP and the Notch precursor (which is the expected
substrate for presenilin-dependent S3 cleavage). In contrast, PAMP
did not bind to NICD, which arises as a product of
presenilin-PAMP-mediated S3 cleavage of the Notch precursor.
Example 4
PAMP Screening of Schizophrenia Patients
[0145] A study is conducted to investigate PAMP sequence, its
expression levels, and its activity, in selected study objects.
Initially, the study objects are selected from families having (1)
increased rates of schizophrenia, and (2) a high proportion linked
to the susceptibility locus on chromosome 1 q21-q22 as described in
Brzustowicz et al., 2000. Control individuals are selected from
families with no or only a rare occurrence of schizophrenia.
[0146] Tissue samples are collected from study objects and control
objects. The samples can be obtained either by sampling tissue
fluids such as blood and cerebrospinal fluid, or by taking biopsies
from selected tissues. In certain instances it may be preferable to
collect tissue biopsies, e.g. from brain, kidney, or lung, from
deceased study or control objects, i.e. post-mortem.
[0147] The samples are analyzed for at least one of the following:
(1) sequence of the entire or selected portions of the PAMP gene;
(2) sequence and levels of PAMP mRNA; (3) sequence, levels, and
activity of PAMP protein; (4) levels of a PAMP substrate.
Identification of relevant mutations in the PAMP gene or mRNA is
performed by using PCR together with primers specific for PAMP DNA
or mRNA and radiolabeled nucleotides, hybridization analysis,
and/or other automated sequencing techniques described herein or in
references provided in the present disclosure, which are all
incorporated by reference. Mutations in and levels of the PAMP
protein is studied by, e.g., purifying PAMP from the tissue sample,
performing enzyme-linked immunosorbent assay (ELISA) or other
quantitative or semi-quantitative immunoassays, Edman degradation
analysis, mass-spectroscopy, Western blotting, or other analytical
techniques described herein or in references in the present
disclosure. PAMP biological activity assays are conducted as
described herein by either in vivo methods (e.g., monitoring
.beta.APP processing and the production of amyloid-.beta. peptide
(A.beta.), or other suitable protein substrates for PAMP including
Notch, etc.), or by in vitro assays (using either whole cell or
cell-free assays to measure processing of suitable substrates
including .beta.APP or parts thereof, and other proteins such as
Notch).
[0148] The results from these assays will preferably show any
significant correlation between mutations in and/or expression
levels of PAMP or the PAMP gene and susceptibility to
schizophrenia. PAMP or PAMP mutations, or altered PAMP or PAMP
levels, identified in this manner can advantageously be used in the
creation of in vivo assays (e.g., transgenic animals) or in vitro
assays to study induction and/or progression of schizophrenia, as
well as in the screening of potential therapeutic agents for
schizophrenia. For instance, in an in vivo transgenic/recombinant
mouse model, partial phenotypes could be examined via behavioral
deficits in, e.g., exploratory behavior, novelty seeking, cognitive
flexibility/rigidity, sensitivity to dopamine-induced motor
disturbances, etc. (see Cloninger et al., 1996).
[0149] The present invention is not to be limited in scope by the
specific embodiments described herein. Indeed, various
modifications of the invention in addition to those described
herein will become apparent to those skilled in the art from the
foregoing description and the accompanying figures. Such
modifications are intended to fall within the scope of the appended
claims.
[0150] It is further to be understood that values are approximate,
and are provided for description.
[0151] Patents, patent applications, and publications are cited
throughout this application, the disclosures of which are
incorporated herein by reference in their entireties for all
purposes.
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PATENT LITERATURE
[0281] [0282] Canadian Patent Application No. 2,012,311 [0283]
European Patent Publication No. EP 453242 [0284] European Patent
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5,637,684
Sequence CWU 1
1
19 1 1 000 2 2 000 3 422 DNA mouse 3 cccagcggag aggcaagatg
gctacgacta ggggcggctc tgggcctgac ccaggaagtc 60 ggggtcttct
tcttctgtct ttttccgtgg tactggcagg attgtgtggg ggaaactcag 120
tggagaggaa aatctacatt cccttaaata aaacagctcc ttgtgtccgc ctgctcaacg
180 ccactcatca gattggctgc cagtcttcaa ttagtgggga tacaggggtt
atccatgtag 240 tggagaaaga agaagactga agtgggtgtt gacgatggcc
ccaaccccct tacatggtct 300 gctggaggga agtcttcaca gagatgtaat
ggagaagctg aggacaacag tagatcctgg 360 tcttgccgtg attagcagcc
actcacttaa gtttctctag tgtgagtgcc aatgatgggt 420 tt 422 4 473 DNA
unknown EST from unknown organism 4 tggagaggaa aatctacatt
cccttaaata aaacagctcc ttgtgtccgc ctgctcaacg 60 ccactcatca
gattggctgc cagtcttcaa ttagtgggga tacaggggtt atccatgtag 120
tggagaaaga agaagacctg aagtgggtgt tgaccgatgg ccccaacccc ccttacatgg
180 ttctgctgga gggcaagctc ttcaccagag atgtaatgga gaagctgaag
ggaacaacca 240 gtagaatcgc tggtcttgcc gtgactctag ccaagcccaa
ctcaacttca agcttctctc 300 ctagtgtgca gtgcccaaat gatgggtttg
gtaattactc caactcctac gggccagagt 360 ttgctcactg gaagaaaaca
ctgtggaatg aactcggcaa aggcttggct tatgaagacc 420 ttagtttccc
caatcttcct cctggagatg aggaccgaaa caaggtcatc aag 473 5 null 5 000 6
463 DNA unknown EST from unknown organism 6 gggctcgaaa catctctggc
gtggtcctgg ctgaccactc tggctccttc cacaatcggt 60 attaccagag
catttatgac acggctgaga acattaatgt gacctatcct gagtggcaga 120
gccatgaaga ggacctcaac tttgtgacag acactgccaa ggcactggcg aatgtggcca
180 cagtgctggc gcgtgcactg tatgagcttg caggaggaac caacttcagc
agctccatcc 240 aggctgatcc ccagacagtt acacgtctgc tctatgggtt
cctggttaga gctaacaact 300 catggtttca gtcgatcctg aaacatgacc
taaggtccta tttggatgac aggcctcttc 360 aacactacat cgccgtctcc
agccctacca acacgactta cgttgtgcag tacgccttgg 420 caaacctgac
tgggcaaggc gaccaacctc acccgagagc agt 463 7 481 DNA unknown EST from
unknown organism 7 gaggacctca actttgtgac agacactgcc aaggcactgg
cgaatgtggc cacagtgctg 60 gcgcgtgcac tgtatgagct tgcaggagga
accaacttca gcagctccat ccaggctgat 120 ccccagacag ttacacgtct
gctctatggg ttcctggtta gagctaacaa ctcatggttt 180 cagtcgatct
tgaaacatga cctaaggtcc tatttggatg acaggcctct tcaacactac 240
atcgccgtct ccagccctac caacacgact tacgttgtgc agtacgcctt ggcaaacctg
300 actggcaagg cgaccaacct cacccgagag cagtgccagg atccaagtaa
agtcccaaat 360 gagagcaagg atttatatga atactcgtgg gtacaaggcc
cttggaattc caacaggaca 420 gagaggctcc cacagtgtgt gcgctcacag
tgcgactggc aagggcttgt ccctgccttt 480 g 481 8 398 DNA unknown EST
from unknown organism 8 agagctaaca actcatggtt tcagtcgatc ttgaaacatg
acctaaggcc tatttggatg 60 acaggcctct tcaacactac atcgccgtct
ccagccctac caacacgact tacgttgtgc 120 agtacgcctt ggaaacctga
ctggcaaggc gaccaacctc acccgagagc agtgccagga 180 tccaagtaaa
gtcccaaatg agagcaagga tttatatgaa tactcgtggg tacaaggccc 240
ttggaattcc aacaggacag agaggctccc acagtgtgtg cgctccacag tgcgactggc
300 cagggccttg tcccctgcct ttgaactgag tcagtggagc tccacagaat
actctacgtg 360 ggcggagagc cgctggaaag acatccaagc tcggatat 398 9 172
DNA unknown EST from unknown organism 9 tgtgcgctcc acagtgcgac
tggccagggc gttgtcacct gcctttgaac tgagtcagtg 60 gagctccaca
gaatactcta cgtgggcgga gagcgcgtgg aaagacatcc cagctcggat 120
attcctaatt gccagcaaag agcttgagtt catcacgctg atcgtgggct tc 172 10
425 DNA unknown EST from unknown organism 10 tttttttttt ttttttgtat
tgcataattt taatgaaact tgctatttat atacttacaa 60 aaaaaaaaaa
aaaggaaaaa accccaacaa aaatagataa ttatagttta ataataaaaa 120
gtacaactga gcactgtggg ctggaggtgg gatacccact taacagcgtg cccacactaa
180 catgccatct gcacacctgg agaaaggaca gtgggaaaga gacactggct
cagccaggga 240 atccatttct tccctaaggg ttcagggtag ttgaatgcag
atgcacaatc tttcacaccc 300 tcttcctggt gcagcaggtg gctgaatatg
ggggaggggt gtcgggtgac agtggagtca 360 gagggcagta cagggcagga
tggaaggaca gaaggtatcc cgagaaaggg cagaggaggg 420 tgggt 425 11 4560
DNA C. Elegans 11 attaagaacg aatgagtcga tagaagcact gaaaaatgaa
gaaatggcta gttatagtat 60 taattatcgc tggaatacga tgcgacggat
tttcggatca agttttccga actctgttca 120 ttggagaagg aaatgcgtgc
tatagaactt ttaataaaac gcacgaattc ggttgtcaag 180 gtaaaaattg
aatgatttca aataattaca tataaaaaaa tattgcactg ttttttcatt 240
attttcattg aaaattagtg tcaaaatatg tataaatcaa tatttatctg aaaataactg
300 gaaatataga gaaagtgctc caaaatggcc aaaacgttgt caattgccga
agacgatact 360 ctataataaa cggcaattgg caactttcgg gctgtttttc
aacactgttc aatttgtcag 420 atgaaaataa ttttattttc agttaactca
agtgattttc tatattgtgg cagtgaaaaa 480 aattcatagg ccattttgta
gaattgccga aaataactcc acctctgaat tacatgcatt 540 ttcactagaa
aatatcattt acatacattt taatttataa atatccagta tttatttatt 600
ttcttaaact cattttcaag aaaaatattt tcagctaatc gagaaaacga gaatggccta
660 attgttcgaa tcgacaaaca ggaagacttc aaaaatctcg attcttgctg
gaattcattt 720 tatcccaaat attccgggaa atattgggca cttctcccag
tcaatttgat tcgtcgtgat 780 acaatttctc aattgaaatc atcgaaatgt
ctttctggaa tagtattata taatagtgga 840 gaatctattc atccaggaga
tgaatcaaca gcagcttcac atgatgcaga atgtccaaat 900 gctgcaagtg
attattatct tcaagataaa aatgaagaat attgtgaaag aaagattaat 960
tctcggggtg ctataacacg agatggatta atgaaaattg attggcggat acaaatggta
1020 tttattgata attcaactga tttggaaatc attgagaaat gttattcaat
gttcaataaa 1080 ccaaaagaag atggttcatc tggatatcca tattgtggaa
tgagctttcg tttggctaat 1140 atggcggctg gaaattcaga aatttgctat
cggcgtggaa aaaacgatgc aaagctgttt 1200 cagatgaata ttgatagcgg
gtaggttttt aaattttaag cagttaaaag aggtgaattt 1260 ttgcattatt
aaatgcagaa tagaccgtaa atattgcatg atgagatgta tttcatgata 1320
atattcttta agaaaataaa tttgaaaatt tcataggaaa ataaacaaaa ttttgctaaa
1380 cttcatagtt tggcatttct tatctcgttt tttgttaatt taggggattt
tttagtcaat 1440 aattgcaccg attccatgta tctctttttt tcgaatgata
ttgtacctat atgccagacg 1500 agctataatt tcctaatttt aaaaaataaa
ttgtccaact caatgcctca atagttgaag 1560 ttttccagag atgctcctca
actctgtggt gcaatgcaca gtgacaatat atttgcattt 1620 ccaactccaa
ttccaacttc tccaacaaat gagacaataa tcacgagcaa atatatgatg 1680
gtaactgctc gaatggacag ttttggaatg attccagaga tttctgttgg cgaagtatcc
1740 gtactaactt caattatttc tgtactcgca gcagctcgat caatgggaac
acagatcgaa 1800 aaatggcaga aagcatcgaa tacttcgaat cgtaatgttt
tctttgcttt tttcaatggt 1860 gaatcgttgg attatattgg aagtggtgcg
gctgcgtatc agatggagta agttggaaaa 1920 tttaatttaa aaaacgttct
agaactagta actgatcaaa aaaatttccc tattaacata 1980 aaatggccca
aaaattccta aaaatttcaa aatttcaaaa aaaaaaatag ttcgggcaaa 2040
aaacataatt ctagctgaaa cctcaaattt ggcaagcttt tcaggctcgt aacatatttt
2100 tggaagtcgt caatcaaaaa ataattcagt tttattcatt tatgataatt
aattaaaatt 2160 ttccaacatt gtttgaaaat ttttataatg atatttggtc
attttaccat aattggaatg 2220 gttttcaatt attttcccac tcttccttta
gagaaaaaat atatttgtct tcagaaatgg 2280 aaagttccca caaatgattc
gctctgatcg aacacacatt catccaattc gcccgaatga 2340 gttagattat
atactggaag tacaacaaat tggagttgct aaaggacgaa aatattatgt 2400
acacgttgat ggagaacgat atcaacagaa taagacacag acagatcgag ttattgatcg
2460 aattgaacga ggtcttcgta gtcatgcttt tgatcttgaa aaaccatctg
gaagtggaga 2520 taggtgggtg catcgaaaat agtttttttt ttcaagaaca
tacagaaaac gaaaagcttt 2580 taaagcattt tctttaaaaa ttaaaacaat
ttgagcatat gtaaactaca attccgagtg 2640 tcgtttttcg aaaaaagtct
aaaattaaaa aaaagcttat cgctcactat ttttcgaaaa 2700 taaggtattt
ttcctttaat aaaggcaaac gaaaaatctt cagccatgga taggtgaatt 2760
atagaaataa ttttcaaaaa ttttcctttt tcagagttcc acccgcaagt tggcactcgt
2820 ttgccaaggc tgatgctcac gttcaatcag ttctccttgc accatatggt
aaagaatatg 2880 aatatcaacg agttaattca attttggata aaaatgagtg
gacagaagac gaacgagaga 2940 aagcaattca agagattgaa gctgtttcta
ctgctattct ggcagcagcc gctgattatg 3000 ttggagttga aactgatgaa
gttgttgcaa aagttgataa aaaattggta tgtattcttt 3060 ttttttttaa
ttttaaaact ttcagcgaca atttagatgt tttattgttg aatttgaaat 3120
ttgcagtatt tttaaatact taaaacaaaa tccctgatga cgcagcgatt catcgctgta
3180 ttttctaatt gctgaaattg aattccatat atatggaata tttcatatct
ttacatataa 3240 acgttttttt ttttcagata accactatat tcgattgtct
catcacttcc aatttctggt 3300 tcgactgtga ttttatgcaa aaactcgatg
gcggtcgcta ccacaagctg tttaattcct 3360 acggttttaa tcaaaaatca
acatatattt caatggaatc ccatactgca ttccctaccg 3420 tactccattg
gttaactatt ttcgctttgg gtagtgacaa agaaacattg aatgtgaaaa 3480
gtgaaaagag ctgctcacat cttggtcaat ttcaagcggt gagtttttat tttaaacgaa
3540 tatcaaataa ttaaaatagt tttccgccag tttcagatgt atacctacac
gtggcaaccg 3600 aatccgtaca ccggaaattt cagttgtctg aaatctgcaa
ttgttaaaaa agtaatggtt 3660 tcgccggctg tagattctca aacacccgaa
gaagaaatga acacgagata ttcaacatgg 3720 atggaatcag tttatattat
tgaatctgtg aatttatatt tgatggaaga tgcttcattt 3780 gaatatacaa
tgattctgat tgcggttatt tctgctttat tatcaatctt tgcagttggt 3840
tagttttttt ttcaaaaaaa aaattacaaa aataaatcac aagctttcga gctttctcgt
3900 attcgaaaat gaaggagttt cgcattaaag aaaactagat tttgaatcag
tttttctaat 3960 ctttagagaa attatactca catttgatgc ccagaaaagt
ttgcgacttt tgagccaaaa 4020 gcacggtgcc aggtctcgac acgaaaaatt
tatattaatt gaaaatatgt ttgcgccttt 4080 aaatggtact gtattttcga
attctcattg ctggcgattt aaaaaaatgc attttttaaa 4140 tccataaaag
ttgagaaaaa tcgatgaaaa attgcacaga aatgagtgca agaaattaca 4200
gtattcttta aaggcgcaca ccttttcgca tttcacaaaa tttcatcgtg tcgataccgg
4260 gtaccgtatt ttggaggcaa aaatcgcaaa atctcgcgtc tggataatat
cgtttatcgt 4320 ttattgaagg aagtttttaa aaataagaaa aattgacagc
tgcgagaaat tatgcataat 4380 ttataaaaca ataaaaattt tttttttcag
gtcgctgttc tgaaacaaca tttatcgttg 4440 acgagggaga accagcagcg
gaaggaggag aacctcttta acaaattatt ctcttcaaca 4500 atgtatcata
aattgattaa tttatttaat atttatattc gaaaaaatgt tcccattttt 4560 12 721
PRT C. Elegans 12 Met Lys Lys Trp Leu Val Ile Val Leu Ile Ile Ala
Gly Ile Arg Cys 1 5 10 15 Asp Gly Phe Ser Asp Gln Val Phe Arg Thr
Leu Phe Ile Gly Glu Gly 20 25 30 Asn Ala Cys Tyr Arg Thr Phe Asn
Lys Thr His Glu Phe Gly Cys Gln 35 40 45 Ala Asn Arg Glu Asn Glu
Asn Gly Leu Ile Val Arg Ile Asp Lys Gln 50 55 60 Glu Asp Phe Lys
Asn Leu Asp Ser Cys Trp Asn Ser Phe Tyr Pro Lys 65 70 75 80 Tyr Ser
Gly Lys Tyr Trp Ala Leu Leu Pro Val Asn Leu Ile Arg Arg 85 90 95
Asp Thr Ile Ser Gln Leu Lys Ser Ser Lys Cys Leu Ser Gly Ile Val 100
105 110 Leu Tyr Asn Ser Gly Glu Ser Ile His Pro Gly Asp Glu Ser Thr
Ala 115 120 125 Ala Ser His Asp Ala Glu Cys Pro Asn Ala Ala Ser Asp
Tyr Tyr Leu 130 135 140 Gln Asp Lys Asn Glu Glu Tyr Cys Glu Arg Lys
Ile Asn Ser Arg Gly 145 150 155 160 Ala Ile Thr Arg Asp Gly Leu Met
Lys Ile Asp Trp Arg Ile Gln Met 165 170 175 Val Phe Ile Asp Asn Ser
Thr Asp Leu Glu Ile Ile Glu Lys Cys Tyr 180 185 190 Ser Met Phe Asn
Lys Pro Lys Glu Asp Gly Ser Ser Gly Tyr Pro Tyr 195 200 205 Cys Gly
Met Ser Phe Arg Leu Ala Asn Met Ala Ala Gly Asn Ser Glu 210 215 220
Ile Cys Tyr Arg Arg Gly Lys Asn Asp Ala Lys Leu Phe Gln Met Asn 225
230 235 240 Ile Asp Ser Gly Asp Ala Pro Gln Leu Cys Gly Ala Met His
Ser Asp 245 250 255 Asn Ile Phe Ala Phe Pro Thr Pro Ile Pro Thr Ser
Pro Thr Asn Glu 260 265 270 Thr Ile Ile Thr Ser Lys Tyr Met Met Val
Thr Ala Arg Met Asp Ser 275 280 285 Phe Gly Met Ile Pro Glu Ile Ser
Val Gly Glu Val Ser Val Leu Thr 290 295 300 Ser Ile Ile Ser Val Leu
Ala Ala Ala Arg Ser Met Gly Thr Gln Ile 305 310 315 320 Glu Lys Trp
Gln Lys Ala Ser Asn Thr Ser Asn Arg Asn Val Phe Phe 325 330 335 Ala
Phe Phe Asn Gly Glu Ser Leu Asp Tyr Ile Gly Ser Gly Ala Ala 340 345
350 Ala Tyr Gln Met Glu Asn Gly Lys Phe Pro Gln Met Ile Arg Ser Asp
355 360 365 Arg Thr His Ile His Pro Ile Arg Pro Asn Glu Leu Asp Tyr
Ile Leu 370 375 380 Glu Val Gln Gln Ile Gly Val Ala Lys Gly Arg Lys
Tyr Tyr Val His 385 390 395 400 Val Asp Gly Glu Arg Tyr Gln Gln Asn
Lys Thr Gln Thr Asp Arg Val 405 410 415 Ile Asp Arg Ile Glu Arg Gly
Leu Arg Ser His Ala Phe Asp Leu Glu 420 425 430 Lys Pro Ser Gly Ser
Gly Asp Arg Val Pro Pro Ala Ser Trp His Ser 435 440 445 Phe Ala Lys
Ala Asp Ala His Val Gln Ser Val Leu Leu Ala Pro Tyr 450 455 460 Gly
Lys Glu Tyr Glu Tyr Gln Arg Val Asn Ser Ile Leu Asp Lys Asn 465 470
475 480 Glu Trp Thr Glu Asp Glu Arg Glu Lys Ala Ile Gln Glu Ile Glu
Ala 485 490 495 Val Ser Thr Ala Ile Leu Ala Ala Ala Ala Asp Tyr Val
Gly Val Glu 500 505 510 Thr Asp Glu Val Val Ala Lys Val Asp Lys Lys
Leu Ile Thr Thr Ile 515 520 525 Phe Asp Cys Leu Ile Thr Ser Asn Phe
Trp Phe Asp Cys Asp Phe Met 530 535 540 Gln Lys Leu Asp Gly Gly Arg
Tyr His Lys Leu Phe Asn Ser Tyr Gly 545 550 555 560 Phe Asn Gln Lys
Ser Thr Tyr Ile Ser Met Glu Ser His Thr Ala Phe 565 570 575 Pro Thr
Val Leu His Trp Leu Thr Ile Phe Ala Leu Gly Ser Asp Lys 580 585 590
Glu Thr Leu Asn Val Lys Ser Glu Lys Ser Cys Ser His Leu Gly Gln 595
600 605 Phe Gln Ala Met Tyr Thr Tyr Thr Trp Gln Pro Asn Pro Tyr Thr
Gly 610 615 620 Asn Phe Ser Cys Leu Lys Ser Ala Ile Val Lys Lys Val
Met Val Ser 625 630 635 640 Pro Ala Val Asp Ser Gln Thr Pro Glu Glu
Glu Met Asn Thr Arg Tyr 645 650 655 Ser Thr Trp Met Glu Ser Val Tyr
Ile Ile Glu Ser Val Asn Leu Tyr 660 665 670 Leu Met Glu Asp Ala Ser
Phe Glu Tyr Thr Met Ile Leu Ile Ala Val 675 680 685 Ile Ser Ala Leu
Leu Ser Ile Phe Ala Val Gly Arg Cys Ser Glu Thr 690 695 700 Thr Phe
Ile Val Asp Glu Gly Glu Pro Ala Ala Glu Gly Gly Glu Pro 705 710 715
720 Leu 13 2949 DNA human 13 tctgcagaat tcggcttgcg cctggaaaca
cgaacttccg gtctcttagg ctccgggcca 60 cagagacggt gtcagtggta
gcctagagag gccgctaaca gacaggagcc gaacgggggc 120 ttccgctcag
cagagaggca agatggctac ggcagggggt ggctctgggg ctgacccggg 180
aagtcggggt ctccttcgcc ttctgtcttt ctgcgtccta ctagcaggtt tgtgcagggg
240 aaactcagtg gagaggaaga tatatatccc cttaaataaa acagctccct
gtgttcgcct 300 gctcaacgcc actcatcaga ttggctgcca gtcttcaatt
agtggagaca caggggttat 360 ccacgtagta gagaaagagg aggacctaca
gtgggtattg actgatggcc ccaacccccc 420 ttacatggtt ctgctggaga
gcaagcattt taccagggat ttaatggaga agctgaaagg 480 gagaaccagc
cgaattgctg gtcttgcagt gtccttgacc aagcccagtc ctgcctcagg 540
cttctctcct agtgtacagt gcccaaatga tgggtttggt gtttactcca attcctatgg
600 gccagagttt gctcactgca gagaaataca gtggaattcg ctgggcaatg
gtttggctta 660 tgaagacttt agtttcccca tctttcttct tgaagatgaa
aatgaaacca aagtcatcaa 720 gcagtgctat caagatcaca acctgagtca
gaatggctca gcaccaacct tcccactatg 780 tgccatgcag ctcttttcac
acatgcatgc tgtcatcagc actgccacct gcatgcggcg 840 cagctccatc
caaagcacct tcagcatcaa cccagaaatc gtctgtgacc ccctgtctga 900
ttacaatgtg tggagcatgc taaagcctat aaatacaact gggacattaa agcctgacga
960 cagggttgtg gttgctgcca cccggctgga tagtcgttcc tttttctgga
atgtggcccc 1020 aggggctgaa agcgcagtgg cttcctttgt cacccagctg
gctgctgctg aagctttgca 1080 aaaggcacct gatgtgacca ccctgccccg
caatgtcatg tttgtcttct ttcaagggga 1140 aacttttgac tacattggca
gctcgaggat ggtctacgat atggagaagg gcaagtttcc 1200 cgtgcagtta
gagaatgttg actcatttgt ggagctggga caggtggcct taagaacttc 1260
attagagctt tggatgcaca cagatcctgt ttctcagaaa aatgagtctg tacggaacca
1320 ggtggaggat ctcctggcca cattggagaa gagtggtgct ggtgtccctg
ctgtcatcct 1380 caggaggcca aatcagtccc agcctctccc accatcttcc
ctgcagcgat ttcttcgagc 1440 tcgaaacatc tctggcgttg ttctggctga
ccactctggt gccttccata acaaatatta 1500 ccagagtatt tacgacactg
ctgagaacat taatgtgagc tatcccgaat ggctgagccc 1560 tgaagaggac
ctgaactttg taacagacac tgccaaggcc ctggcagatg tggccacggt 1620
gctgggacgt gctctgtatg agcttgcagg aggaaccaac ttcagcgaca cagttcaggc
1680 tgatccccaa acggttaccc gcctgctcta tgggttcctg attaaagcca
acaactcatg 1740 gttccagtct atcctcaggc aggacctaag gtcctacttg
ggtgacgggc ctcttcaaca 1800 ttacatcgct gtctccagcc ccaccaacac
cacttatgtt gtacagtatg ccttggcaaa 1860 tttgactggc acagtggtca
acctcacccg agagcagtgc caggatccaa gtaaagtccc 1920 aagtgaaaac
aaggatctgt atgagtactc atgggtccag ggccctttgc attctaatga 1980
gacggaccga ctcccccggt gtgtgcgttc tactgcacga ttagccaggg ccttgtctcc
2040 tgcctttgaa ctgagtcagt ggagctctac tgaatactct acatggactg
agagccgctg 2100 gaaagatatc cgtgcccgga tatttctcat cgccagcaaa
gagcttgagt tgatcaccct 2160 gacagtgggc ttcggcatcc tcatcttctc
cctcatcgtc acctactgca tcaatgccaa 2220 agctgatgtc cttttcattg
ctccccggga gccaggagct gtgtcatact gagsaggacc 2280 scagcttttc
ttgccagctc agcagttcac ttcctagagc atctgtccca ctgggacaca 2340
accactaatt tgtcactgga acctccctgg gcctgtctca gattgggatt aacataaaag
2400 agtggaacta tccaaaagag acagggagaa ataaataaat tgcctccctt
cctccgctcc 2460 cctttcccat caccccttcc ccatttcctc ttccttctct
actcatgcca gattttggga 2520 ttacaaatag aagcttcttg ctcctgttta
actccctagt tacccaccct aatttgccct 2580 tcaggaccct tctacttttt
ccttcctgcc ctgtacctct ctctgctcct cacccccacc 2640 cctgtaccca
gccaccttcc tgactgggaa ggacataaaa ggtttaatgt cagggtcaaa 2700
ctacattgag cccctgagga caggggcatc tctgggctga gcctactgtc tccttcccac
2760 tgtcctttct ccaggccctc agatggcaca ttagggtggg cgtgctgcgg
gtgggtatcc 2820 cacctccagc ccacagtgct cagttgtact ttttattaag
ctgtaatatc tatttttgtt 2880 tttgtctttt tcctttattc tttttgtaaa
tatatatata atgagtttca ttaaaataga 2940 ttatcccac 2949 14 709 PRT
human 14 Met Ala Thr Ala Gly Gly Gly Ser Gly Ala Asp Pro Gly Ser
Arg Gly 1 5 10 15 Leu Leu Arg Leu Leu Ser Phe Cys Val Leu Leu Ala
Gly Leu Cys Arg 20 25 30 Gly Asn Ser Val Glu Arg Lys Ile Tyr Ile
Pro Leu Asn Lys Thr Ala 35 40 45 Pro Cys Val Arg Leu Leu Asn Ala
Thr His Gln Ile Gly Cys Gln Ser 50 55 60 Ser Ile Ser Gly Asp Thr
Gly Val Ile His Val Val Glu Lys Glu Glu 65 70 75 80 Asp Leu Gln Trp
Val Leu Thr Asp Gly Pro Asn Pro Pro Tyr Met Val 85 90 95 Leu Leu
Glu Ser Lys His Phe Thr Arg Asp Leu Met Glu Lys Leu Lys 100 105 110
Gly Arg Thr Ser Arg Ile Ala Gly Leu Ala Val Ser Leu Thr Lys Pro 115
120 125 Ser Pro Ala Ser Gly Phe Ser Pro Ser Val Gln Cys Pro Asn Asp
Gly 130 135 140 Phe Gly Val Tyr Ser Asn Ser Tyr Gly Pro Glu Phe Ala
His Cys Arg 145 150 155 160 Glu Ile Gln Trp Asn Ser Leu Gly Asn Gly
Leu Ala Tyr Glu Asp Phe 165 170 175 Ser Phe Pro Ile Phe Leu Leu Glu
Asp Glu Asn Glu Thr Lys Val Ile 180 185 190 Lys Gln Cys Tyr Gln Asp
His Asn Leu Ser Gln Asn Gly Ser Ala Pro 195 200 205 Thr Phe Pro Leu
Cys Ala Met Gln Leu Phe Ser His Met His Ala Val 210 215 220 Ile Ser
Thr Ala Thr Cys Met Arg Arg Ser Ser Ile Gln Ser Thr Phe 225 230 235
240 Ser Ile Asn Pro Glu Ile Val Cys Asp Pro Leu Ser Asp Tyr Asn Val
245 250 255 Trp Ser Met Leu Lys Pro Ile Asn Thr Thr Gly Thr Leu Lys
Pro Asp 260 265 270 Asp Arg Val Val Val Ala Ala Thr Arg Leu Asp Ser
Arg Ser Phe Phe 275 280 285 Trp Asn Val Ala Pro Gly Ala Glu Ser Ala
Val Ala Ser Phe Val Thr 290 295 300 Gln Leu Ala Ala Ala Glu Ala Leu
Gln Lys Ala Pro Asp Val Thr Thr 305 310 315 320 Leu Pro Arg Asn Val
Met Phe Val Phe Phe Gln Gly Glu Thr Phe Asp 325 330 335 Tyr Ile Gly
Ser Ser Arg Met Val Tyr Asp Met Glu Lys Gly Lys Phe 340 345 350 Pro
Val Gln Leu Glu Asn Val Asp Ser Phe Val Glu Leu Gly Gln Val 355 360
365 Ala Leu Arg Thr Ser Leu Glu Leu Trp Met His Thr Asp Pro Val Ser
370 375 380 Gln Lys Asn Glu Ser Val Arg Asn Gln Val Glu Asp Leu Leu
Ala Thr 385 390 395 400 Leu Glu Lys Ser Gly Ala Gly Val Pro Ala Val
Ile Leu Arg Arg Pro 405 410 415 Asn Gln Ser Gln Pro Leu Pro Pro Ser
Ser Leu Gln Arg Phe Leu Arg 420 425 430 Ala Arg Asn Ile Ser Gly Val
Val Leu Ala Asp His Ser Gly Ala Phe 435 440 445 His Asn Lys Tyr Tyr
Gln Ser Ile Tyr Asp Thr Ala Glu Asn Ile Asn 450 455 460 Val Ser Tyr
Pro Glu Trp Leu Ser Pro Glu Glu Asp Leu Asn Phe Val 465 470 475 480
Thr Asp Thr Ala Lys Ala Leu Ala Asp Val Ala Thr Val Leu Gly Arg 485
490 495 Ala Leu Tyr Glu Leu Ala Gly Gly Thr Asn Phe Ser Asp Thr Val
Gln 500 505 510 Ala Asp Pro Gln Thr Val Thr Arg Leu Leu Tyr Gly Phe
Leu Ile Lys 515 520 525 Ala Asn Asn Ser Trp Phe Gln Ser Ile Leu Arg
Gln Asp Leu Arg Ser 530 535 540 Tyr Leu Gly Asp Gly Pro Leu Gln His
Tyr Ile Ala Val Ser Ser Pro 545 550 555 560 Thr Asn Thr Thr Tyr Val
Val Gln Tyr Ala Leu Ala Asn Leu Thr Gly 565 570 575 Thr Val Val Asn
Leu Thr Arg Glu Gln Cys Gln Asp Pro Ser Lys Val 580 585 590 Pro Ser
Glu Asn Lys Asp Leu Tyr Glu Tyr Ser Trp Val Gln Gly Pro 595 600 605
Leu His Ser Asn Glu Thr Asp Arg Leu Pro Arg Cys Val Arg Ser Thr 610
615 620 Ala Arg Leu Ala Arg Ala Leu Ser Pro Ala Phe Glu Leu Ser Gln
Trp 625 630 635 640 Ser Ser Thr Glu Tyr Ser Thr Trp Thr Glu Ser Arg
Trp Lys Asp Ile 645 650 655 Arg Ala Arg Ile Phe Leu Ile Ala Ser Lys
Glu Leu Glu Leu Ile Thr 660 665 670 Leu Thr Val Gly Phe Gly Ile Leu
Ile Phe Ser Leu Ile Val Thr Tyr 675 680 685 Cys Ile Asn Ala Lys Ala
Asp Val Leu Phe Ile Ala Pro Arg Glu Pro 690 695 700 Gly Ala Val Ser
Tyr 705 15 2250 DNA mouse 15 cccagcggag aggcaacatg gctacgacta
ggggcggctc tgggcctgac ccaggaagtc 60 ggggtcttct tcttctgtct
ttttccgtgg tactggcagg attgtgtggg ggaaactcag 120 tggagaggaa
aatctacatt cccttaaata aaacagctcc ttgtgtccgc ctgctcaacg 180
ccactcatca gattggctgc cagtcttcaa ttagtgggga tacaggggtt atccacgtag
240 tggagaaaga agaagacctg aagtgggtgt tgaccgatgg ccccaacccc
ccttacatgg 300 ttctgctgga gggcaagctc ttcaccagag atgtaatgga
gaagctgaag ggaacaacca 360 gtagaatcgc tggtcttgcc gtgactctag
ccaagcccaa ctcaacttca agcttctctc 420 ctagtgtgca gtgcccaaat
gatgggtttg gtatttactc caactcctac gggccagagt 480 ttgctcactg
caagaaaaca ctgtggaatg aactgggcaa cggcttggct tatgaagact 540
ttagtttccc catctttctt cttgaagatg agaacgaaac caaggtcatc aagcagtgct
600 atcaagatca caacctgggt cagaatggct ctgcaccaag cttcccattg
tgtgctatgc 660 agctcttctc acacatgcac gccgtcatca gcactgccac
ctgcatgcgg cgcagcttca 720 tccagagcac cttcagcatc aacccagaaa
tcgtctgtga ccccttatct gactacaacg 780 tatggagcat gcttaagcct
ataaatacat ctgtgggact agaacctgac gtcagggttg 840 tggttgcggc
cacacggctg gatagccggt cctttttctg gaatgtggcc ccaggggctg 900
aaagtgctgt agcctccttt gtcactcagc tggctgcagc tgaagctttg cacaaggcac
960 ctgatgtgac cactctatcc cgaaatgtga tgtttgtctt cttccagggg
gaaacttttg 1020 actacattgg cagctcacgg atggtctatg atatggagaa
cggcaagttt cccgtgcggc 1080 tcgagaacat cgactccttc gtggagctgg
gacaggtggc cctaagaact tcactagatc 1140 tctggatgca cacagatccc
atgtctcaga aaaatgagtc tgtgaaaaac caggtggagg 1200 atcttctggc
cactctggag aagagcggtg ctggtgtccc tgaagttgtc ctgaggagac 1260
tggcccagtc ccaggccctt ccaccttcat ctctacaacg atttcttcgg gctcgaaaca
1320 tctctggcgt ggtcctggct gaccactctg gctccttcca caatcggtat
taccagagca 1380 tttatgacac ggctgagaac attaatgtga cctatcctga
gtggcagagc ccagaagagg 1440 acctcaactt tgtgacagac actgccaagg
cactggcgaa tgtggccaca gtgctggcgc 1500 gtgcactgta tgagcttgca
ggaggaacca acttcagcag ctccatccag gctgatcccc 1560 agacagttac
acgtctgctc tatgggttcc tggttagagc taacaactca tggtttcagt 1620
cgatcctgaa acatgaccta aggtcctatt tggatgacag gcctcttcaa cactacatcg
1680 ccgtctccag ccctaccaac acgacttacg ttgtgcagta cgccttggca
aacctgactg 1740 gcaaggcgac caacctcacc cgagagcagt gccaggatcc
aagtaaagtc ccaaatgaga 1800 gcaaggattt atatgaatac tcgtgggtac
aaggcccttg gaattccaac aggacagaga 1860 ggctcccaca gtgtgtgcgc
tccacagtgc gactggccag ggccttgtcc cctgcctttg 1920 aactgagtca
gtggagctcc acagaatact ctacgtgggc ggagagccgc tggaaagaca 1980
tccaagctcg gatattccta attgccagca aagagcttga gttcatcacg ctgatcgtgg
2040 gcttcagcac ccttgtcttc tctctcatcg tcacctactg catcaatgcc
aaagccgacg 2100 tccttttygt tgctccccga gagccaggag ctgtgtctta
ctgaagagga ctctagctct 2160 ccctgcctgc tctgaacttt acttcccaga
ccaggtgtcc ggctgggaac aaaccactaa 2220 tttgtcactg gactgtctct
gggcctgctt 2250 16 708 PRT mouse 16 Met Ala Thr Thr Arg Gly Gly Ser
Gly Pro Asp Pro Gly Ser Arg Gly 1 5 10 15 Leu Leu Leu Leu Ser Phe
Ser Val Val Leu Ala Gly Leu Cys Gly Gly 20 25 30 Asn Ser Val Glu
Arg Lys Ile Tyr Ile Pro Leu Asn Lys Thr Ala Pro 35 40 45 Cys Val
Arg Leu Leu Asn Ala Thr His Gln Ile Gly Cys Gln Ser Ser 50 55 60
Ile Ser Gly Asp Thr Gly Val Ile His Val Val Glu Lys Glu Glu Asp 65
70 75 80 Leu Lys Trp Val Leu Thr Asp Gly Pro Asn Pro Pro Tyr Met
Val Leu 85 90 95 Leu Glu Gly Lys Leu Phe Thr Arg Asp Val Met Glu
Lys Leu Lys Gly 100 105 110 Thr Thr Ser Arg Ile Ala Gly Leu Ala Val
Thr Leu Ala Lys Pro Asn 115 120 125 Ser Thr Ser Ser Phe Ser Pro Ser
Val Gln Cys Pro Asn Asp Gly Phe 130 135 140 Gly Ile Tyr Ser Asn Ser
Tyr Gly Pro Glu Phe Ala His Cys Lys Lys 145 150 155 160 Thr Leu Trp
Asn Glu Leu Gly Asn Gly Leu Ala Tyr Glu Asp Phe Ser 165 170 175 Phe
Pro Ile Phe Leu Leu Glu Asp Glu Asn Glu Thr Lys Val Ile Lys 180 185
190 Gln Cys Tyr Gln Asp His Asn Leu Gly Gln Asn Gly Ser Ala Pro Ser
195 200 205 Phe Pro Leu Cys Ala Met Gln Leu Phe Ser His Met His Ala
Val Ile 210 215 220 Ser Thr Ala Thr Cys Met Arg Arg Ser Phe Ile Gln
Ser Thr Phe Ser 225 230 235 240 Ile Asn Pro Glu Ile Val Cys Asp Pro
Leu Ser Asp Tyr Asn Val Trp 245 250 255 Ser Met Leu Lys Pro Ile Asn
Thr Ser Val Gly Leu Glu Pro Asp Val 260 265 270 Arg Val Val Val Ala
Ala Thr Arg Leu Asp Ser Arg Ser Phe Phe Trp 275 280 285 Asn Val Ala
Pro Gly Ala Glu Ser Ala Val Ala Ser Phe Val Thr Gln 290 295 300 Leu
Ala Ala Ala Glu Ala Leu His Lys Ala Pro Asp Val Thr Thr Leu 305 310
315 320 Ser Arg Asn Val Met Phe Val Phe Phe Gln Gly Glu Thr Phe Asp
Tyr 325 330 335 Ile Gly Ser Ser Arg Met Val Tyr Asp Met Glu Asn Gly
Lys Phe Pro 340 345 350 Val Arg Leu Glu Asn Ile Asp Ser Phe Val Glu
Leu Gly Gln Val Ala 355 360 365 Leu Arg Thr Ser Leu Asp Leu Trp Met
His Thr Asp Pro Met Ser Gln 370 375 380 Lys Asn Glu Ser Val Lys Asn
Gln Val Glu Asp Leu Leu Ala Thr Leu 385 390 395 400 Glu Lys Ser Gly
Ala Gly Val Pro Glu Val Val Leu Arg Arg Leu Ala 405 410 415 Gln Ser
Gln Ala Leu Pro Pro Ser Ser Leu Gln Arg Phe Leu Arg Ala 420 425 430
Arg Asn Ile Ser Gly Val Val Leu Ala Asp His Ser Gly Ser Phe His 435
440 445 Asn Arg Tyr Tyr Gln Ser Ile Tyr Asp Thr Ala Glu Asn Ile Asn
Val 450 455 460 Thr Tyr Pro Glu Trp Gln Ser Pro Glu Glu Asp Leu Asn
Phe Val Thr 465 470 475 480 Asp Thr Ala Lys Ala Leu Ala Asn Val Ala
Thr Val Leu Ala Arg Ala 485 490 495 Leu Tyr Glu Leu Ala Gly Gly Thr
Asn Phe Ser Ser Ser Ile Gln Ala 500 505 510 Asp Pro Gln Thr Val Thr
Arg Leu Leu Tyr Gly Phe Leu Val Arg Ala 515 520 525 Asn Asn Ser Trp
Phe Gln Ser Ile Leu Lys His Asp Leu Arg Ser Tyr 530 535 540 Leu Asp
Asp Arg Pro Leu Gln His Tyr Ile Ala Val Ser Ser Pro Thr 545 550 555
560 Asn Thr Thr Tyr Val Val Gln Tyr Ala Leu Ala Asn Leu Thr Gly Lys
565 570 575 Ala Thr Asn Leu Thr Arg Glu Gln Cys Gln Asp Pro Ser Lys
Val Pro 580 585 590 Asn Glu Ser Lys Asp Leu Tyr Glu Tyr Ser Trp Val
Gln Gly Pro Trp 595 600 605 Asn Ser Asn Arg Thr Glu Arg Leu Pro Gln
Cys Val Arg Ser Thr Val 610 615 620 Arg Leu Ala Arg Ala Leu Ser Pro
Ala Phe Glu Leu Ser Gln Trp Ser 625 630 635 640 Ser Thr Glu Tyr Ser
Thr Trp Ala Glu Ser Arg Trp Lys Asp Ile Gln 645 650 655 Ala Arg Ile
Phe Leu Ile Ala Ser Lys Asp Leu Glu Phe Ile Thr Leu 660 665 670 Ile
Val Gly Phe Ser Thr Leu Val Phe Ser Leu Ile Val Thr Tyr Cys 675 680
685 Ile Asn Ala Lys Ala Asp Val Leu Phe Val Ala Pro Arg Glu Pro Gly
690 695 700 Ala Val Ser Tyr 705 17 2942 DNA D. melanogaster 17
tctgatatca tcgccactgt gctgggaatt cggcacgagc gcaacactgc aatttctgga
60 cgcgatttcg tgggaatctt cgatggaaat gcgtctgaat gcggcttcca
tatggctgtt 120 aatactgtcg tatggagcaa ctattgctca aggagaaaga
acccgcgata agatgtacga 180 gcccattgga ggagctagct gtttccgacg
gctgaatggc acccatcaga caggctgttc 240 ctcaacctac tccggttccg
tgggcgtact acatctaata aacgtcgagg ccgacctgga 300 atttcttctt
agcagcccac catctccacc ttacgccccc atgataccac ctcacctgtt 360
cacacgtaac aacctgatgc gcctaaagga agccggacca aagaacattt ctgtggtgct
420 gctgatcaac cgcacgaacc agatgaagca gttctcgcac gaactcaact
gccccaatca 480 gtacagcggc ctgaacagca ccagtgagac ctgcgacgcc
agcaatccag ccaaaaactg 540 gaatccctgg ggcactggac ttctgcacga
ggactttccc tttcctatct attacatagc 600 cgatttggat caggtcacca
agctagagaa gtgctttcag gactttaaca accataacta 660 cgagacgcac
gcgctgcgta gcttgtgcgc cgtcgaggtc aagtccttta tgtccgccgc 720
tgtcaacacc gaggtctgta tgcgccgcac caacttcatc aataatcttg gaggaagcaa
780 gtactgcgat ccgctcgagg gacggaatgt ttcgccacct tgtacccccg
aaagccagca 840 atcggaaaca actttggaga cagtccatac gaatgaaaag
ttcatattag taacctgtcg 900 cctggacacc accaccatgt tcgatggcgt
cggtcttgga gccatggact ccctcatggg 960 atttgctgtt ttcactcatg
tggcgtatct attaaaacaa ctacttccgc cgcaaagcaa 1020 agaccttcat
aatgtcctct ttgtgacttt taatggcgaa tcctatgact acattggttc 1080
tcaaagattt gtatacgaca tggagaaact tcaatttcct actgaatcca caggcacgcc
1140 tccgattgcc tttgacaata ttgacttcat gctggacatc gggacactgg
atgacatatc 1200 gaatattaag ctgcatgcgt taaatggaac gactttggct
cagcaaattc tagagcggct 1260 aaataactat gcgaagtcgc cacgctatgg
ctttaacctg aacattcagt ccgagatgag 1320 cgctcactta ccacctacgt
cggcgcaatc atttctgcga cgtgatccaa acttcaatgc 1380 attgattcta
aacgctcgtc caacgaacaa gtattatcat tccacatacg atgacgcgga 1440
taacgtggac ttcacctatg cgaacacaag caaggatttc acccagctga cggaagttaa
1500 tgactttaaa agcttgaacc cagattcact gcaaatgaaa gtgcgcaacg
tttcctctat 1560 tgtggccatg gccctatatc agacaataac tggaaaggag
tacactggca ccaaggtggc 1620 caaccctctg atggcagatg agttccttta
ctgtttcctg caatcggcgg actgcccact 1680 ctttaaggcc gcatcttatc
cgggcagtca gctcaccaat ttgcctccga tgcgctacat 1740 aagcgtcttg
ggtggctctc aagagtcgtc gggctatacg tatagattgc tgggctatct 1800
cttgtcacaa ctgcagccag acattcacag agataactgc accgacttgc cgctgcacta
1860 tttcgccgga ttcaacaata tcggagagtg tcgcctcacc acgcagaact
acagtcacgc 1920 cctgagtcca gcttttctta ttgatggcta cgattggagt
tccggcatgt attccacttg 1980 gactgaatcc acctggtcac agttcagtgc
acgcatcttc ctgcgcccgt ccaatgtgca 2040 ccaggtcaca actctaagcg
ttggcatagt ggtgctgata atatccttct gtttggtgta 2100 tataatcagc
tcacgatcgg aagtcctctt tgaggatttg ccggcaagca atgccgcatt 2160
atttggttga tgtcacaact gccacagcga cgaaatcatc gcgttcagca gctcattcca
2220 tagatttctg catgcgtaaa ctaaacgtta cttgtaaacc aatcgattaa
gaatttctga 2280 ttgtgccctt ttagatcgcc gcggccgaca gcctgttaaa
tcttcaaaga atatctgatc 2340 acgtgccgaa gatgattggt tgctgaatat
gatctaaaac aaaaaacaga cttaacaaag 2400 acactaaaat gatattctaa
ctcgtcttat ttaaaacatt aagcaaacgt ttatttatat 2460 gtatttttgt
attttaaagt agtaaattag cttctatcaa ctacgttgta tcatatatag 2520
acatttaacc aattgcgaca aaattctttc cacttgtccg gccctttttg cattgtacat
2580 aggattaacc aacccaattg aacctctgat aatgccaagg aagagatgtc
tgtacaaata 2640 ttgacaagaa actgctataa cttataaatc actggaaata
tttatacctt ctgcatctat 2700 tgcgatactg aacttaatga tctgaaatca
ttacttcata gaagacaaat aattattata 2760 acgactttaa attatatatg
tttaataaat tttgataagg tgtaaagcaa tgtcctgtta 2820 tctagttagg
ttattttcaa ggcaattatt cacagctctc agattccaac gattgatgta 2880
gtttaatctc aactctttac caaagaagtc catttgtact agtgtaaaag atattttcaa
2940 ta 2942 18 695 PRT D. melanogaster 18 Met Glu Met Arg Leu Asn
Ala Ala Ser Ile Trp Leu Leu Ile Leu Ser 1 5 10 15 Tyr Gly Ala Thr
Ile Ala Gln Gly Glu Arg Thr Arg Asp Lys Met Tyr 20 25 30 Glu Pro
Ile Gly Gly Ala Ser Cys Phe Arg Arg Leu Asn Gly Thr His 35 40 45
Gln Thr Gly Cys Ser Ser Thr Tyr Ser Gly Ser Val Gly Val Leu His 50
55 60 Leu Ile Asn Val Glu Ala Asp Leu Glu Phe Leu Leu Ser Ser Pro
Pro 65 70 75 80 Ser Pro Pro Tyr Ala Pro Met Ile Pro Pro His Leu Phe
Thr Arg Asn 85 90 95 Asn Leu Met Arg Leu Lys Glu Ala Gly Pro Lys
Asn Ile Ser Val Val
100 105 110 Leu Leu Ile Asn Arg Thr Asn Gln Met Lys Gln Phe Ser His
Glu Leu 115 120 125 Asn Cys Pro Asn Gln Tyr Ser Gly Leu Asn Ser Thr
Ser Glu Thr Cys 130 135 140 Asp Ala Ser Asn Pro Ala Lys Asn Trp Asn
Pro Trp Gly Thr Gly Leu 145 150 155 160 Leu His Glu Asp Phe Pro Phe
Pro Ile Tyr Tyr Ile Ala Asp Leu Asp 165 170 175 Gln Val Thr Lys Leu
Glu Lys Cys Phe Gln Asp Phe Asn Asn His Asn 180 185 190 Tyr Glu Thr
His Ala Leu Arg Ser Leu Cys Ala Val Glu Val Lys Ser 195 200 205 Phe
Met Ser Ala Ala Val Asn Thr Glu Val Cys Met Arg Arg Thr Asn 210 215
220 Phe Ile Asn Asn Leu Gly Gly Ser Lys Tyr Cys Asp Pro Leu Glu Gly
225 230 235 240 Arg Asn Val Ser Pro Pro Cys Thr Pro Glu Ser Gln Gln
Ser Glu Thr 245 250 255 Thr Leu Glu Thr Val His Thr Asn Glu Lys Phe
Ile Leu Val Thr Cys 260 265 270 Arg Leu Asp Thr Thr Thr Met Phe Asp
Gly Val Gly Leu Gly Ala Met 275 280 285 Asp Ser Leu Met Gly Phe Ala
Val Phe Thr His Val Ala Tyr Leu Leu 290 295 300 Lys Gln Leu Leu Pro
Pro Gln Ser Lys Asp Leu His Asn Val Leu Phe 305 310 315 320 Val Thr
Phe Asn Gly Glu Ser Tyr Asp Tyr Ile Gly Ser Gln Arg Phe 325 330 335
Val Tyr Asp Met Glu Lys Leu Gln Phe Pro Thr Glu Ser Thr Gly Thr 340
345 350 Pro Pro Ile Ala Phe Asp Asn Ile Asp Phe Met Leu Asp Ile Gly
Thr 355 360 365 Leu Asp Asp Ile Ser Asn Ile Lys Leu His Ala Leu Asn
Gly Thr Thr 370 375 380 Leu Ala Gln Gln Ile Leu Glu Arg Leu Asn Asn
Tyr Ala Lys Ser Pro 385 390 395 400 Arg Tyr Gly Phe Asn Leu Asn Ile
Gln Ser Glu Met Ser Ala His Leu 405 410 415 Pro Pro Thr Ser Ala Gln
Ser Phe Leu Arg Arg Asp Pro Asn Phe Asn 420 425 430 Ala Leu Ile Leu
Asn Ala Arg Pro Thr Asn Lys Tyr Tyr His Ser Thr 435 440 445 Tyr Asp
Asp Ala Asp Asn Val Asp Phe Thr Tyr Ala Asn Thr Ser Lys 450 455 460
Asp Phe Thr Gln Leu Thr Glu Val Asn Asp Phe Lys Ser Leu Asn Pro 465
470 475 480 Asp Ser Leu Gln Met Lys Val Arg Asn Val Ser Ser Ile Val
Ala Met 485 490 495 Ala Leu Tyr Gln Thr Ile Thr Gly Lys Glu Tyr Thr
Gly Thr Lys Val 500 505 510 Ala Asn Pro Leu Met Ala Asp Glu Phe Leu
Tyr Cys Phe Leu Gln Ser 515 520 525 Ala Asp Cys Pro Leu Phe Lys Ala
Ala Ser Tyr Pro Gly Ser Gln Leu 530 535 540 Thr Asn Leu Pro Pro Met
Arg Tyr Ile Ser Val Leu Gly Gly Ser Gln 545 550 555 560 Glu Ser Ser
Gly Tyr Thr Tyr Arg Leu Leu Gly Tyr Leu Leu Ser Gln 565 570 575 Leu
Gln Pro Asp Ile His Arg Asp Asn Cys Thr Asp Leu Pro Leu His 580 585
590 Tyr Phe Ala Gly Phe Asn Asn Ile Gly Glu Cys Arg Leu Thr Thr Gln
595 600 605 Asn Tyr Ser His Ala Leu Ser Pro Ala Phe Leu Ile Asp Gly
Tyr Asp 610 615 620 Trp Ser Ser Gly Met Tyr Ser Thr Trp Thr Glu Ser
Thr Trp Ser Gln 625 630 635 640 Phe Ser Ala Arg Ile Phe Leu Arg Pro
Ser Asn Val His Gln Val Thr 645 650 655 Thr Leu Ser Val Gly Ile Val
Val Leu Ile Ile Ser Phe Cys Leu Val 660 665 670 Tyr Ile Ile Ser Ser
Arg Ser Glu Val Leu Phe Glu Asp Leu Pro Ala 675 680 685 Ser Asn Ala
Ala Leu Phe Gly 690 695 19 17 PRT human 19 Ala Arg Leu Ala Arg Ala
Leu Ser Pro Ala Phe Glu Leu Ser Gln Trp 1 5 10 15 Ser
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