U.S. patent application number 11/125970 was filed with the patent office on 2006-07-20 for mammalian neuralized family transcriptional regulators and uses.
This patent application is currently assigned to CeMines, Inc.. Invention is credited to Kaia Palm, Tonis Timmusk.
Application Number | 20060160103 11/125970 |
Document ID | / |
Family ID | 25198619 |
Filed Date | 2006-07-20 |
United States Patent
Application |
20060160103 |
Kind Code |
A1 |
Palm; Kaia ; et al. |
July 20, 2006 |
Mammalian neuralized family transcriptional regulators and uses
Abstract
The disclosure relates to isolated polynucleotides and purified
polypeptides of the Neu family of proteins, which have been shown
to demonstrate transcriptional regulatory activity. For example,
the purified polynucleotide can encode a Neu polypeptide, wherein
the Neu polypeptide comprises at least one neuralized homology
repeat domain and a C3HC4 RING-zinc finger domain is disclosed. A
purified Neu polypeptide, wherein the Neu polypeptide comprises at
least one neuralized homology repeat domain and a C3HC4 RING-zinc
finger domain is disclosed. Antibodies capable of specifically
binding to the disclosed Neu polypeptides are disclosed. Vectors
expressing the disclosed Neu protein coding regions and host cells
containing the vectors are disclosed. Methods of making the Neu
proteins disclosed are also provided, as are method of identifying
binding partners that interact with a Neu protein family
member.
Inventors: |
Palm; Kaia; (Tallinn,
EE) ; Timmusk; Tonis; (Helsinki, FI) |
Correspondence
Address: |
Todd A. Lorenz;DORSEY & WHITNEY LLP
Intellectual Property Department
555 California Street, Suite 1000
San Francisco
CA
94104-1513
US
|
Assignee: |
CeMines, Inc.
|
Family ID: |
25198619 |
Appl. No.: |
11/125970 |
Filed: |
May 9, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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09808387 |
Mar 14, 2001 |
6962985 |
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11125970 |
May 9, 2005 |
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Current U.S.
Class: |
435/6.14 ;
435/320.1; 435/325; 435/455; 435/69.1; 530/399; 536/23.5 |
Current CPC
Class: |
A61P 35/00 20180101;
C07K 14/82 20130101; A61K 38/00 20130101; C07K 14/71 20130101 |
Class at
Publication: |
435/006 ;
435/069.1; 435/320.1; 435/325; 435/455; 530/399; 536/023.5 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C07H 21/04 20060101 C07H021/04; C12P 21/06 20060101
C12P021/06; C07K 14/475 20060101 C07K014/475 |
Claims
1-30. (canceled)
31. A purified neuralized (Neu) polypeptide, comprising at least
one neuralized homology repeat domain and a C3HC4 RING-zinc finger
domain, wherein said Neu polypeptide has at least 90% sequence
identity to SEQ ID NO:22 and is capable of suppressing
transcription.
32. The purified Neu polypeptide according to claim 31, wherein
said Neu polypeptide has at least 95% sequence identify to SEQ ID
NO:22.
33. The purified Neu polypeptide according to claim 31, wherein
said Neu polypeptide comprises SEQ ID NO:22.
34. The purified Neu polypeptide according to claim 31, wherein
said neuralized homology repeat domain comprises SEQ ID NO:48.
35. An antibody capable of specifically binding to an isolated Neu
polypeptide according to claim 31.
36. The antibody according to claim 35, wherein said antibody
specifically binds to a polypeptide comprising at least 10
consecutive amino acids of said Neu polypeptide.
37. The antibody according to claim 35, wherein said antibody is a
monoclonal antibody.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The described invention relates to the family of mammalian
Neuralized (neu) genes, proteins encoded by those genes (Neu),
expression patterns of the gene family, their function as a
transcriptional regulator, and the proteins with which the Neu
family of proteins interacts. Additionally, therapeutic and
diagnostic uses for the Neu family of proteins and agents that bind
thereto are also provided.
[0003] 2. Description of the Related Art
[0004] Development and functioning of the nervous system requires
orchestrated action of thousands of transcriptional regulators.
Balance between transcriptional activators and repressors
determines the spectrum of expressed genes. The molecular basis of
the initial stages of neurogenesis as well as several aspects of
neuronal differentiation have been extensively studied. As a
result, a variety of transcriptional activators and repressors have
been discovered and characterized. Our understanding of these
systems and their component interactions, however, is far from
complete. Little is known about the molecular mechanisms that
support neuronal circuits in developing and mature nervous systems
or the molecular mechanisms that coordinate maintenance of the
differentiated state.
[0005] The process of lateral inhibition prevents neighboring cells
from developing into the same type of differentiated cells in flies
and in vertebrates. Tanabe & Jessell, Science, 274:1115-23
(1996). In Drosophila, a group of mutations has been described that
shows severe defects in the process of lateral inhibition in the
developing nervous system. These neurogenic mutations result in
hyperplasia of the neural tissue at the expense of epidermal
structures. Campos-Ortega & Jan, Annu Rev Neurosci, 14:399-420
(1991). The temporal and spatial expression patterns of the neu are
compatible with its function as a neurogenic gene in Drosophila.
The Neu protein is expressed throughout the ectoderm at the time
when cell fate is determined and its expression proceeds in
neuroblasts. Boulianne, et al., EMBO J, 10:2975-2983 (1991). neu
expression has been detected in actively proliferating neuroblasts
in several regions of the CNS and PNS. Expression of neu in
imaginal disc suggests that it is also involved in later stages of
development. The neu gene encodes a RING finger (C3HC4) type zinc
finger protein. The molecular function of the Drosophila Neu
protein is unknown. Interestingly, it was discovered that EST data
bases contain a homologue of Neu suggesting that a family of
Neu-like proteins is present in Drosophila.
[0006] Current studies of the brain development in Drosophila and
vertebrates, indicate that many basic molecular and genetic
mechanisms involved in neurogenesis are highly conserved. During
development of the nervous system, neural cell specification is
acquired through the series of progressive restrictive steps. In
Drosophila, neural precursors are first specified by proneural
genes including basic helix-loop-helix (bHLH) transcription factors
of atonal and achaete-scute complex. Simpson, Neuron, 15:739-742
(1995). The process of lateral inhibition, which further restricts
the developmental potential of neuroectodermal cells is regulated
by neurogenic genes such as Notch, mastermind, big brain, Delta,
Enhancer of split, and neuralized. Analysis of the function of
these neurogenic loci in the Drosophila embryo has revealed that
mutations in any of these genes result in hyperplasia of neural
tissue at the expense of epidermal structures and also cause
defects in tissues derived from mesoderm and endoderm.
Campos-Ortega and Jan, Annu. Rev Neurosci, 14:399-420 (1991);
Harentstein et al., Development, 116:1203-1220 (1992). Vertebrate
homologues of Notch, Delta and the proneural/neurogenic genes of
atonal, achaete-scute, hairy, and Enhancer of Split complex have
been identified and recent work, mostly in Xenopus and mouse,
suggests that their role in neurogenesis is conserved. Lewis, Curr
Opin Neurobiol, 6:3-10 (1996); Kageyama et al., Int J Biochem Cell
Biol, 29:1389-1399 (1997); and Beatus, Lendahl, J Neurosci Res
54:125-136 (1998). For example, postnatal Notch signaling affects
the elaboration of different body systems and regulates plasticity
of cortical postmitotic neurons. Artavanis-Tsakonas et al.,
Science, 284:770-776 (1999); Redmond et al., Nat Neurosci, 3:30-40
(2000); and Sestan et al., Science, 286:741-746 (1999).
[0007] The last few years have brought the identification and
characterization of many new key regulators of vertebrate
neurogenesis. Recently, a human homologue of Drosophila Neu gene
was isolated and its expression in the adult nervous system and in
tumors of neuroectodermal origin, such as astrocytomas, was
characterized. Nakamura, et al., Oncogene 16(8):1009-1019 (1998).
Nakamura and others (1998) hypothesized that h-neu1 plays a role in
determination of cell fate in the central nervous system and may
act as a tumor suppressor which inactivation could be associated
with malignant progression of astrocytic tumors. A homology search
in human, rat, and mouse EST databases revealed three new mammalian
Neu homologs, suggesting that a family of Neu-like proteins exists
in mammals.
SUMMARY OF THE INVENTION
[0008] The disclosure relates to isolated polynucleotides and
purified polypeptides of the Neu family of proteins, which have
been shown to demonstrate transcriptional regulatory activity. For
example, the purified polynucleotide can encode a Neu polypeptide,
wherein the Neu polypeptide comprises at least one neuralized
homology repeat domain and a C3HC4 RING-zinc finger domain is
disclosed. A purified Neu polypeptide, wherein the Neu polypeptide
comprises at least one neuralized homology repeat domain and a
C3HC4 RING-zinc finger domain is disclosed. Antibodies capable of
specifically binding to the disclosed Neu polypeptides are
disclosed. Vectors expressing the disclosed Neu protein coding
regions and host cells containing the vectors are disclosed.
Methods of making the Neu proteins disclosed are also provided, as
are methods of identifying binding partners that interact with a
Neu protein family member.
BRIEF DESCRIPTION OF THE DRAWINGS
[0009] FIG. 1 shows the primary structure of neu1 protein isoforms
in mouse and rat.
[0010] FIG. 2 is an analysis of neu1 mRNA expression by RNase
protection assay in mouse and rat.
[0011] FIG. 3 is an in situ hybridization analysis of neu1 mRNA
expression in the developing and adult mouse brain.
[0012] FIG. 4 is an in situ hybridization analysis of neu1 mRNA
expression in adult rat brain.
[0013] FIG. 5 shows expression of neu1 mRNA in an adult rat nervous
system.
[0014] FIG. 6 shows cellular localization of neu1 mRNA in an adult
rat nervous system.
[0015] FIG. 7 shows the combined analyses of in situ hybridization
of neu1 mRNA and immunohistochemistry of the neuronal marker
neuN.
[0016] FIG. 8 shows neu1 mRNA expression in the hippocampus of
adult rat brain after kainic acid treatment.
[0017] FIG. 9 shows transcriptional analysis from various promoters
in transient expression assays.
[0018] FIG. 10 shows that neuralized homology repeat domains of
neu1 mediate the transcriptional repression when fused to the DNA
binding domain of Gal4.
[0019] FIG. 11 shows the subcellular localization of neu-FLAG and
neu-EGFP fusion proteins in Neuro2A cells, using FLAG
immunofluorescence using anti-FLAG antibody (A, E, F, G) and direct
fluorescence of EGFP fusion proteins (B, C, D, H).
[0020] FIG. 12 shows alignment of neuralized homology repeat
domains of human Neu1, Neu2, and Neu3 proteins.
[0021] FIG. 13 shows the analysis of neu2 mRNA expression by RNase
protection assay in mouse and rat.
[0022] FIG. 14 shows the analysis of neu3 mRNA expression by RNase
protection in mouse.
[0023] FIG. 15 is an analysis of the expression of rat Neu1
interactors by RNase protection assay.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0024] The invention described herein relates to the identification
of members of the Neuralized (neu) family of genes and their
respective proteins, and aspects of their structure and function as
transcriptional regulators. The invention relates to the
characteristics of mammalian neu gene family's conserved structural
motifs, related function as transcriptional regulators, and
functional features of the family relating to expression patterns
and potential interactions with interaction-partners, providing
tools with which to explore the Neu-related regulatory cascades
that operate in neurons and other cell types.
Structure
[0025] The invention described herein relates to the neuralized
(Neu) family of transcriptional regulators that contain a
C-terminal C3HC4 RING zinc finger and at least one neuralized
homology repeat domain. The term "neuralized" (or "Neu"), as used
herein, includes all members of the Neu family such as Neu1, Neu2,
Neu3, Neu4 coding sequences and proteins. These proteins belong to
a family of proteins that share C-terminal C3HC4 RING zinc finger
and at least one neuralized homology repeat domain. Neuralized
homology repeat domains represent a novel class of transcription
repression domains that regulate transcription of a large number of
genes.
[0026] The C3HC4 RING-zinc finger motif is a cystine-rich amino
acid sequence motif found in the sequence of the human RING gene.
Freemont, et al., Biochem J, 278:1-23 (1991); Freemont, Ann NY Acad
Sci, 684:174-192 (1993). The motif can be described as
C--X.sub.2--C--X.sub.(9-27)--C--X.sub.(1-3)--H--X.sub.2--C--X.sub.2--C--X-
.sub.(4-48)--C--X.sub.2--C, where C is a cysteine, H is a
histamine, and X can be any amino acid. This family includes genes
that are involved in the regulation of development,
differentiation, apoptosis, oncogenesis, and membrane
trafficking.
[0027] Several isolated and characterized RING-zinc finger proteins
have proposed roles in gene regulation. Although the precise
function of the RING-zinc finger domain is unknown, indirect data
demonstrates that the RING-zinc finger domain could function as a
DNA-binding or protein-protein interaction domain. These two
functions are related. Several transcriptional repressors which act
as dimers have been characterized. Their structures comprise both
dimerization and DNA-binding surfaces. For example, well known
transcriptional regulators that contain RING-zinc finger domains
are BRCA1 (Miki et al., Science, 266(5182):66-71 (1994)) and MEL18
(Kanno et al., EMBO J. 15;14(22):5672-8 (1995)), a polycomb
group-related transcriptional regulator.
Function and Interactions
[0028] Neu Function as a Transcriptional Regulator
[0029] Mammalian Neu1 acts as a powerful transcriptional repressor
in transient expression assays and silences both, TATA-containing
and TATA-less promoters, including the promoters of NGF, BDNF,
NF-L, and GAP-43. NHRs function as transcription repression
domains, suggesting that NHR-containing proteins represent a novel
class of transcriptional repressors. It is likely that mammalian
Neu1 mediates transcriptional repression through protein-protein
interactions. Like several known repressors, mammalian Neu1 could
function through interaction with co-repressors such as dCtBP or
mammalian homologues of Groucho, and general repressor complexes,
such as NC2, Mot1, or Not to interfere with the function of Pol II
complex. Maldonado et al., Cell, 99:455-458 (1999) and Mannervik et
al., Science, 284:606-609 (1999). Alternatively, repression could
be achieved through chromatin remodeling by recruiting the histone
deacetylase complexes (HDACs). Glass and Rosenfeld, Endocr Rev,
21:447 (2000); Knoepfler and Eisenman, Cell, 99:447-450 (1999); and
Torchia et al., Curr Opin Cell Biol, 10:373-383 (1998). Current
data suggest that the mechanism of Neu1 repression does not include
a HDAC complex in neuroblastoma Neuro2A cells, as the HDAC
inhibitor trichostatinA did not relieve m-Neu1-mediated repression
in these cells.
[0030] Neu is a Shuttling Protein with Dominant Cytoplasmic
Localization as a Result of a Nuclear Import Combined with an
Efficient Export
[0031] A putative nuclear localization signal has been identified
in the N-terminus of d-Neu, however, the NLS sequence identified in
d-Neu, is not conserved in mouse, rat and human Neu proteins.
Boulianne et al., EMBO J 10:2975-2983 (1991) and Price et al., EMBO
J, 12:2411-2418 (1993). The weak NLS sequences (HKAVKR (SEQ ID NO:
43), RLKITKK (SEQ ID NO: 44)), that were identified in mammalian
Neu1 proteins, have been suggested to regulate nuclear import of a
fraction of the synthesized protein. Boulikas, J Cell Biochem,
60:61-82 (1996). Indeed, m-Neu1 resides both in the cytoplasm and
in the nucleus, revealing that it is the subject of regulated
nuclear import. Recent studies have shown that importin-.alpha.
family members are involved in the formation of the NLS receptor
complexes that govern the protein transport to the nucleus. Ullman
et al., Cell, 90:967-970 (1997); and Izaurralde and Adam, RNA,
4:351-364 (1998). Interestingly, importin-.alpha.3 was identified
here as one of the m-Neu interacting proteins by yeast two-hybrid
screening. Furthermore, the results discussed herein also
demonstrate that the CRM1/exportin1-related export pathway controls
the nucleocytoplasmic shuttling of Neu1, since the nuclear export
of a tagged-m-Neu1 fusion protein is blocked by LMB. LMB is a
microbial metabolite that inactivates the nuclear export by
interfering with the binding of CRM1/exportin1 to the nuclear
export signals. Fornerod et al., Cell, 90:1051-1060 (1997); Kudo et
al., Exp Cell Res, 242:540-547 (1998); Kudo et al., Proc Natl Acad
Sci USA, 96:9112-9117 (1999); Fukuda et al., Nature, 390:308-311
(1997); and Nishi et al., J Biol Chem, 269:6320-6324 (1994). These
data reveal that mammalian Neu1 function is additionally regulated
by nucleocytoplasmic shuttling.
[0032] Neu Function as a Calcium-Signal Transducer
[0033] Transcriptional activity of Neu is controlled by calcium
(Ca.sup.2+) signaling that regulates Neu translocation into the
nucleus where Neu acts as a transcriptional repressor. It has been
long known that intracellular calcium controls a variety of brain-
and muscle cell functions. Since Ca.sup.2+ levels regulate
nucleocytoplasmic shuttling of transcription factors (Crabtree,
Cell, 96:611-614 (1999)), Neu proteins could function as mediators
of calcium signaling to the nucleus to regulate gene expression.
There is evidence that in Drosophila and also in vertebrates,
ligand activated Notch is subjected to proteolytic cleavage and
transported to the nucleus where it acts as a transcriptional
regulator (Jarriault et al 1995; Ohtsuka et al., 1999).
Interestingly, recently it was shown that d-neu protein is
associated primarily with the plasma membrane (Yeh et al., 2000).
This, however, does not exclude the possibility that Neu1, like
Notch, may be localized also within the nucleus. Based on current
knowledge it is hypothesized that Neu1 functions as a mediator of
an extracellular signal from the plasma membrane to the nucleus to
regulate gene expression. According to one possible scenario, Neu1
is posttranslationally modified in a signal (Ca.sup.2+)-dependent
fashion and subsequently translocated into the nucleus, where it
functions as a transcriptional regulator.
[0034] Neurogenic Function of Neu.
[0035] Drosophila neuralized (d-neu) and h-neu1 genes encode
homologous (.about.40%) proteins with a C-terminal C3HC4 RING zinc
finger domain (RZD) and one or two neuralized homology repeat (NHR)
domains. Boulianne et al., EMBO J, 10:2975-2983 (1991) and Price et
al., EMBO J, 12:2411-2418 (1993). d-neu is expressed in the
ectoderm at the time when cell fate is determined, implying the
role of Neu in neurogenesis. Boulianne et al., EMBO J, 10:2975-2983
(1991).
[0036] In Drosophila, d-neu expression has been detected earliest
in the ectoderm continuing later in neuroblasts. Boulianne et al.,
EMBO J. 10:2975-2983 (1991). Since developing mouse CNS can be
divided into regions that express either high or low levels of
neu1, and as neu1 is not expressed in proliferating regions of the
nervous system, it suggests that other neu-related genes function
in a fashion complementary to neu1. Indeed, based on the recent
findings we could argue that neu1 defines a new gene and protein
family consisting of at least two Drosophila and four mammalian
genes.neu2, second member of mammalian neu family of genes, is
expressed at high levels in the embryonic brain, whereas the
expression levels decrease during postnatal development. Recent
studies of the brain development in Drosophila and vertebrates,
indicate that many basic molecular and genetic mechanisms involved
in neurogenesis are highly conserved. Lewis, J. Curr Opin
Neurobiol, 6:3-10 (1996); Kageyama et al., Int J Biochem Cell Biol,
29:1389-1399 (1997); and Beatus & Lendahl, J Neurosci Res,
54:125-136 (1998). For example, postnatal Notch signaling affects
the elaboration of different body systems and regulates plasticity
of cortical postmitotic neurons. Artavanis-Tsakonas et al.,
Science, 284:770-776 (1999); Redmond et al., Nat Neurosci, 3:30-40
(2000); and Sestan et al., Science, 741-746 (1999).
[0037] Earlier genetic studies in Drosophila suggested that delta,
mastermind, big brain, and neuralized refine a signal upstream of
notch and that mastermind functions upstream of all the other
neurogenic genes. Campos-Ortega and Jan, Annu Rev Neurosci,
14:399-420 (1991); and Lieber, Genes Dev, 7:1949-1965 (1993).
Therefore, if the function of the mammalian Neu family of proteins
is conserved in conjunction with other neurogenic factors,
mammalian neu genes refine the signal upstream of mammalian
homologues of notch and downstream of mastermind in tissues where
neu genes are expressed, particularly in developing nervous system.
Given that mammalian neu mRNAs encode different protein isoforms,
complex regulatory circuits implicating various Neu family members
are expected in different tissues. The transcription repression
activities of mammalian Neu could well-explain its function as a
neurogenic gene.
[0038] Neu Role in Cell Signalling and Synaptogenesis
[0039] Suppression of gene expression plays an important role in
the maintenance and stability of a mature nervous system. It is
essential to suppress neurite growth and extensive formation of new
axons and dendrites in the adult functional nervous system and to
maintain neurons in their differentiated state. Transcriptional
repressors are involved and play a crucial role in the silencing of
the neurite growth program.
[0040] Mammalian neu1 shows most prominent expression in the
postnatal central nervous system, revealing its function in
postnatal development. neu1 mRNA expression levels increase
significantly during the early postnatal development when active
synaptogenesis takes place, to reach the peak levels in the adult
animal. In the adult mammalian CNS, the highest expression levels
of neu1 assign to the neurons of hippocampus, cerebral cortex,
striatum, and amygdala. Whereas several brain regions such as
thalamus/hypothalamus, midbrain, medulla, and also the spinal cord
exhibit low expression. This indicates to the independent
regulation of neu1 expression in various brain regions by specific
signaling mechanisms as well as to the requirement of Neu function
for different cell-cell signaling systems.
[0041] neu1 mRNA expression studies in adult rat brain revealed
that in several brain regions, particularly in the granular cells
of dentate gyrus of hippocampus, neu1 mRNA is localized in the
dendrites, suggesting that synthesis of Neu1 protein also occurs in
dendrites. Several data indicate that proteins locally translated
from dendritic mRNAs at activated synapses provide basis for
activity-dependent regulation of synaptic modulation (reviewed in
Steward et al., Neuron, 21:741-51 (1997); Kuhl and Skehel, Curr
Opin Neurobiol, 8(5):600-6 (1998); Schuman, Neuron, 23:645-8
(1999); Tiedge et al., Science, 283:186-7 (1999); Kiebler and
DesGroseillers, Neuron, 25:19-28 (2000)). Accordingly, Neu could be
involved in the regulation of neuritogenesis and/or synaptogenesis,
affecting the generation of the precise pattern of neuronal
connectivity. Recent molecular perturbation experiments suggested
that Notch1 signalling in cortical neurons promotes dendritic
branching and inhibits neurite growth (Redmond et al., 2000; Sestan
et al., 1999). Suggesting that the function of neu1 to refine a
signal upstream of Notch in Drosophila is evolutionary conserved in
mammalian nervous system, it is possible, that Neu and Notch
pathways act in an interrelated manner to confer developmental
plasticity to adult neurons. The possible similarities in the
molecular mechanisms of function of two genes, Notch and neu1, that
were first discovered as neurogenic, are appealingly apparent.
[0042] Neu Role Related to Repair and Regeneration After Injury to
the CNS
[0043] A specific temporal order of events at the cellular and
molecular level occurs in response to injury to the brain.
Injury-compromised neurons degenerate while surviving neurons
undergo neuritogenesis and synaptogenesis to establish neuronal
connectivity destroyed in the injury. In the brain, after
kainate-induced change in neuronal activity (a neurotoxic,
excitotoxic or ischemic insult), it was observed that a consistent
down-regulation of neu1 mRNA in the hippocampal formation with a
strong reduction in the molecular layer where granule cell
dendrites were present. It is suggested, that after injury of the
CNS, down-regulation of neu1 mRNA expression leads to reduced
levels of Neu1 protein that is essential for derepression of the
transcription of its target genes related to repair and
regeneration, such as growth factors and synaptic proteins.
[0044] Neu Role in Memory and Learning
[0045] Another function of the Neu family of transcription factors
is mediation of Ca.sup.2+ signaling in a variety of neural
processes including learning and memory. A member of Homer family
of proteins, Homer2a, is referred to as an interactor of human Neu1
(GenBank Acc. No. AF081530). Homer proteins are enriched in
excitatory synapses, bind group I metabotropic glutamate receptors
(mGluR), and NMDA receptor interacting Shank proteins and thus can
link NMDA and group I mGluR signaling pathways (reviewed in Xiao et
al., Curr Opin Neurobiol, 10:370-74 (2000)). Given that Homer2a and
Neu1 are co-expressed in various brain structures, it is highly
conceivable that Neu1 participates in the regulation of glutamate
receptor signalling in the adult brain. Glutamate receptor
signaling has been implicated in several forms of
activity-dependent synaptic plasticity, neurodegenerative diseases,
cortical development and addiction. Consequently, if Neu is
involved in glutamate receptor signaling, then the change in Neu
structure, expression or function could lead to various
developmental disorders and mental diseases.
[0046] Thus, it is highly conceivable that neu1 mRNA expression is
regulated by physiological neuronal synaptic activity leading to
reduced levels of Neu1 protein and derepression of the
transcription of its target genes in the processes involving memory
and learning. This function of the Neu family transcription factors
makes them a good target for a variety of drugs that control
different processes in the brain, during development and in
disease. Manipulating this function of Neu can be used to control a
variety of diseases including depression, pain, anxiety, and
neurodegenerative diseases.
[0047] Neu Role in Tumorigenesis
[0048] Also, this invention relates to the role of the Neu family
of factors in the development of tumors, since the human neu1 gene
has been mapped to chromosome 10 within a region which is
frequently deleted in gliomas. neu1 is expressed at varying levels
in different neural and neuroendocrine tumors, including
neuroblastomas, carcinoids, non-small cell lung cancers, and
gliomas, suggesting that expression of Neu family of proteins is
applicable as tumor-specific markers in clinical tumor diagnostics.
Since over-expression of Neu1 blocks DNA synthesis in neuroblastoma
and glioma cells, Neu family of proteins may function also as tumor
suppressor genes.
[0049] Neu Role in Myogenesis and Development of Other Organ
Systems.
[0050] The described herein, especially in the Examples below, show
that expression of neu1 and neu2 is found to be high in at late
embryonic and adult stages of development, also in developing heart
and testes. neu3 was found to be widely expressed, with highest
levels in immune tissues spleen and thymus and in lung. Expression
of neu4 was detected only in muscle and heart. Since Neu proteins
are expressed at various levels in many different body systems, the
role of Neu family of proteins in several developmental pathways is
apparent. Studies of neu.sup.mut flies have reported the
overproduction of nautilus (nau) expressing cells in embryonic and
muscle defects in adult stages of development. Corbin et al., Cell,
67:311-323 (1991); Hartenstein et al., Development, 116:1203-1220
(1992). nau is a myogenic bHLH factor that plays a role in the
differentiation of muscle progenitors in Drosophila. Keller et al.,
Dev Biol, 181:197-212 (1997); Keller et al., Dev Biol, 202:157-171
(1998). Notch signaling has been shown to inhibit MyoD expression
and block myogenesis in mouse. Kuroda et al., J Biol Chem,
274:7238-7244 (1999). It is possible that during mammalian
myogenesis Neu refines Notch signaling by regulating expression of
nau mammalian homologues (MyoD, myf5, myogenin and MRF4).
[0051] Another function of the Neu family of transcription factors
is mediation of Ca.sup.2+ signaling in muscle and other tissues.
This function of the Neu family transcription factors makes them a
good target for a variety of drugs that control different processes
in the muscle, and in those tissues where Neu is expressed.
Manipulating Neu expression and function can be used to control a
variety of diseases including cancer, and muscle-degenerative
diseases, dystrophinopathies, Brody's disease, and malignant
hyperthermia (the last three are caused by the functional
alterations of Ca(2+) signaling).
[0052] Neu Interactions with Neurogenic Genes
[0053] Additionally, activity of transcriptional regulators is
modulated through interactions with other regulatory factors.
Analyses of interactions between Drosophila neurogenic loci has
revealed that neu appears to act upstream of notch (N), enhancer of
split (E(spl)) and delta (Dl), and downstream of mastermind (mam).
Boulianne et al., EMBO J. 10(10), 2975-2983 (1991). Interaction
between neu and E(spl) is observed in the dominant mutation
E(spl)Dl, which is a mutation that enhances the phenotype of split
whereas split is a mutation in the N gene. Enhancement of the split
phenotype increases in the presence of additional copies of neu
gene, but decreases in heterozygotes for neu mutations. Molecular
interactions between these Drosophila neurogenic genes are unknown.
Expression of rat SHARP1 gene (Rossner, et al., Mol Cell Neurosci,
10(3-4):460-475 (1997)), one of the vertebrate homologues of
Drosophila E(spl), is almost identical to the neu1 expression
pattern during development and in adult tissues. The similarity in
expression patterns between neu1 and SHARP1 suggests that products
of these genes could reciprocally affect the function of the other
as it occurs in Drosophila between neu and E(spl) genes.
[0054] Neu Interactions with Proteins Implicated in Nuclear
Transport
[0055] The nucleocytoplasmic transport of functional molecules is
mediated bidirectionally through the nuclear pore complex (NPC),
which spans the double membranes of the nuclear envelope. It has
recently been shown that signaling between the nucleus and the
cytoplasm plays a key role in coordinating the cellular processes
such as the cell cycle and cell differentiation (Yoneda, Cell
Struct Funct 25:205-206. 2000). The weak NLS sequences (HKAVKR (SEQ
ID NO: 43), RLKITKK (SEQ ID NO: 44)), that were identified in
mammalian Neu1 proteins, are indeed, implicated in the regulated
nuclear import of Neu1. Importin-.alpha. family members are
involved in the formation of the NLS receptor complexes that govern
the protein transport to the nucleus. Ullman et al., Cell,
90:967-970 (1997); and Izaurralde and Adam, RNA, 4:351-364 (1998).
We have identified importin-.alpha.3 as one of the m-Neu
interacting proteins by yeast two-hybrid screening.
[0056] Neu Interactions with Miz1/GBP/PIAS Family of Proteins
[0057] Yeast two hybrid screening revealed that Neu1 interacts with
NeuI-1 which is a new splice variant of Miz1/PIASX zinc finger
transcription factor. Miz1 is a sequence specific DNA binding
protein that functions as a positive-acting transcription factor
and interacts directly with homeobox transcription factor Msx2. Wu
et al., 1997 Mech Dev, 65(1-2):3-17 (1997). Msx1 and Msx2, members
of the Msx family of homeobox genes, were found to be important in
inductive tissue interactions. Whereas Msx3 was expressed
exclusively in the developing nervous system. Wang, et al., Mech
Dev, 58(1-2):203-15 (1996). Members of the PIAS family, however,
regulated DNA binding of STAT transcription factors thereby
interfering with the signaling of a variety of cytokines. Chuang,
et al., Biochem Biophys Res Commun, 18;235(2):317-20 (1997) and Liu
et al., Proc Natl Acad Sci USA, 95(18):10626-31 (1998).
[0058] Sequence analyses revealed that PIAS1 is identical to Gu/RNA
helicase II (Gu/RH-II) binding protein GBP. Valdez et al., Biochem
Biophys Res Commun, 234(2):335-40 (1997). The GBP regulates
proteolytic cleavage of Gu/RH-II which could alter its functions or
enzymatic activities or lead to its destruction. These data
indicate that the Neu family of proteins could be involved, perhaps
through its interactions with various proteins, such as the NeuI-1
protein. Such interactions may be involved in several biologically
important regulatory processes including inductive tissue
interactions (Miz1), cytokine signaling (PIAS), and RNA processing
(GBP).
[0059] Neu Interactions with ZNF127 Zinc Finger Family of
Proteins
[0060] Yeast two hybrid screening revealed that Neu1 interacts with
NeuI-2. This protein is a new member of the ZNF127 zinc finger
family of proteins. The ZNF127 gene is localized in
Angelman/Prader-Willi region. Disruption of this gene causes a
genetic defect related to mental retardation. ZNF127, as well as
other genes in this region, were subjected to genomic imprinting
(Mowery-Rushton et al., DNA methylation patterns in human tissues
of uniparental origin using a zinc-finger gene (ZNF127) from the
Angelman/Prader-Willi region Am J Med Genet, 61(2):140-6 (1996)).
The role of ZNF127 in the development of Angelman/Prader-Willi
syndrome, as well as its molecular function, is unknown. The ZNF127
family proteins, however, contain the zinc finger motif,
Cx(8)Cx(5)Cx(3)-H, which is characteristic for viral and early
immediate genes such as TIS11, ERF-2 (Tabara et al., 1999 pos-1
encodes a cytoplasmic zinc-finger protein essential for germline
specification in C. elegans Development, 126(1):1-11); and also to
RNA binding proteins (Carballo et al. Science, 281:1001-1005
(1998)).
[0061] Neu Interactions with Parkin-Like Proteins
[0062] The NeuI-3 protein is another interactor with Neu1 and was
found to be similar to the recently described human gene parkin
(Kitada, et al., Nature 392(6676):605-8 (1998). Mutations in the
parkin gene have been shown to result in autosomal recessive
juvenile parkinsonism. The molecular function of the parkin encoded
protein is unknown. Phylogenetic analysis reveals that ari and
parkin are distant members of a common progeny.
[0063] Neu Interactions with Androgen Receptor Coregulator
ARA54
[0064] NeuI-4, another Neu interactor, is identical to ARA54, an
androgen receptor coregulator (Kang et al., J. Biol. Chem. 274,
8570-8576; 1999). Furthermore, the RING-zinc finger domain of
NeuI-4 has high similarity to Drosophila protein ariadne (ari) that
could be involved in axonal path-finding. Aguilera, et al.,
Genetics 155(3):1231-44 (1996). Two mammalian homologues of ari
have been identified, however, no information is available about
molecular mechanisms of the functioning of the ari family of
proteins.
[0065] Neu Function Based on the Nature of Neu Interactors
[0066] Based on the nature of Neu1 interactors, it is hypothesized
that Neu has the potential to interfere with inductive tissue
interactions (NeuI-1/Miz), cytokine signalling (NeuI-1/PIAS), RNA
processing (NeuI-1/GBP), early immediate responses (NeuI-2/ZNF127),
death of specific cell populations (NeuI-3/parkin), and nuclear
hormone receptor signaling and axonal path-finding
(NeuI-4/ariadne).
Nucleic Acids
[0067] Having identified a number of potential functions for the
Neu family of proteins, the described invention seeks to utilize
this knowledge to manipulate the various developmental pathways in
which Neu functions. As a preliminary step, representative members
of the Neu family of proteins have been isolated and purified.
Polynucleotide molecules encoding the proteins of the Neu family
were then isolated and their sequences are provided below.
[0068] Representative polynucleotide molecules encoding members of
the Neu family include sequences comprising SEQ. ID. NOs.: 1, 3, 5,
7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, and 33.
Polynucleotide molecules encoding Neu family members include those
sequences resulting in minor genetic polymorphisms, differences
between species, and those that contain amino acid substitutions,
additions, and/or deletions.
[0069] In some instances, one can employ such changes in the
sequence of a recombinant Neu to substantially decrease or even
increase the biological activity of Neu relative to the wild-type
Neu activity. Such changes can also be directed towards endogenous
neu sequences using, for example, gene therapy methods to alter the
gene product. Advantageously, the disclosed sequences can be used
to identify and isolate neu polynucleotide encoding molecules from
suitable vertebrate host cells. Thus, in another embodiment, a
method of identifying neu polynucleotide molecules is provided.
[0070] The nucleotide sequences encoding the neuralized homology
repeat domain can be used to identify polynucleotide molecules
encoding other proteins of the Neu family. Complementary DNA
molecules encoding Neu family members can be obtained by
constructing a cDNA library from mRNA of, for example, brain or
muscle tissues that are at different developmental stages. DNA
molecules encoding Neu family members can be isolated from such a
library using the disclosed sequences in standard hybridization
techniques or by amplification of sequences using polymerase chain
reaction (PCR) amplification.
[0071] In a similar manner, genomic DNA encoding Neu can be
obtained using probes designed from the sequences disclosed herein.
Suitable probes for use in identifying Neu family sequences can be
obtained from Neu-specific sequences that are highly conserved
regions between mammalian coding sequences. Primers, for example,
from the neuralized homology motif domains 1 and 2 are suitable for
use in designing PCR primers. Alternatively, oligonucleotides
containing specific DNA sequences from a neu family coding region
can be used to identify related human neu genomic and cDNA clones.
One of skill in the art will appreciate that upstream regulatory
regions of the neu family of genes can be obtained using similar
methods.
[0072] neu family polynucleotide molecules can be isolated using
standard hybridization techniques with probes of at least about 7
nucleotides in length and up to and including the full coding
sequence. Other members of the neu family can be identified using
degenerate oligonucleotides capable of hybridization based on the
sequences disclosed herein for use PCR amplification or by
hybridization at moderate or greater stringency. The term, "capable
of hybridization" as used herein means that the subject nucleic
acid molecules (whether DNA or RNA) anneal to an oligonucleotide of
15 or more contiguous nucleotides of SEQ. ID. NOs.: 1, 3, 5, 7, 9,
11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, and 33.
[0073] The choice of hybridization conditions will be evident to
one skilled in the art and will generally be guided by the purpose
of the hybridization, the type of hybridization (DNA-DNA or
DNA-RNA), and the level of desired relatedness between the
sequences. Methods for hybridization are well established in the
literature. One of ordinary skill in the art realizes that the
stability of nucleic acid duplexes will decrease with an increased
number and location of mismatched bases; thus, the stringency of
hybridization can be used to maximize or minimize the stability of
such duplexes. Hybridization stringency can be altered by:
adjusting the temperature of hybridization; adjusting the
percentage of helix-destabilizing agents, such as formamide, in the
hybridization mix; and adjusting the temperature and salt
concentration of the wash solutions. In general, the stringency of
hybridization is adjusted during the post-hybridization washes by
varying the salt concentration and/or the temperature, resulting in
progressively higher stringency conditions.
[0074] An example of progressively higher stringency conditions is
as follows: 2.times.SSC/0.1% SDS at about room temperature
(hybridization conditions); 0.2.times.SSC/0.1% SDS at about room
temperature (low stringency conditions); 0.2.times.SSC/0.1% SDS at
about 42.degree. C. (moderate stringency conditions); and
0.1.times.SSC at about 68.degree. C. (high stringency conditions).
Washing can be carried out using only one of these conditions,
e.g., high stringency conditions, or each of the conditions can be
used, e.g., for 10-15 minutes each, in the order listed above,
repeating any or all of the steps listed. However, as mentioned
above, optimal conditions will vary, depending on the particular
hybridization reaction involved, and can be determined empirically.
In general, conditions of high stringency are used for the
hybridization of the probe of interest.
[0075] Alternatively, polynucleotides having substantially the same
nucleotide sequence set forth in SEQ. ID. NOs.: 1, 3, 5, 7, 9, 11,
13, 15, 17, 19, 21, 23, 25, 27, 29, 31, and 33 or functional
fragments thereof, or nucleotide sequences that are substantially
identical to SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23,
25, 27, 29, 31, and 33, can represent members of the Neu family of
proteins. By "substantially the same" or "substantially identical"
is meant a nucleic acid or polypeptide exhibiting at least 80%,
85%, 90%, 95% or 100% homology to a reference nucleic acid. For
nucleotide sequences, the length of comparison sequences will
generally be at least 10 to 500 nucleotides in length. More
specifically, the length of comparison will be at least 50
nucleotides, at least 60 nucleotides, at least 75 nucleotides, and
at least 110 nucleotides in length.
[0076] One embodiment of the invention provides isolated and
purified polynucleotide molecules encoding Neu proteins, wherein
the polynucleotide molecules that are capable of hybridizing under
moderate to stringent conditions to an oligonucleotide of 15 or
more contiguous nucleotides of SEQ. ID. NOs.: 1, 3, 5, 7,9, 11, 13,
15, 17, 19, 21, 23, 25, 27, 29, 31, and 33, including complementary
strands thereto.
[0077] DNA sequences of the invention can be obtained by several
methods. For example, the DNA can be isolated using hybridization
or computer-based techniques which are well known in the art. Such
techniques include, but are not limited to: 1) hybridization of
genomic or cDNA libraries with probes to detect homologous
nucleotide sequences; 2) antibody screening of expression libraries
to detect cloned DNA fragments with shared structural features; 3)
polymerase chain reaction (PCR) on genomic DNA or cDNA using
primers capable of annealing to the DNA sequence of interest; 4)
computer searches of sequence databases for similar sequences; and
5) differential screening of a subtracted DNA library.
[0078] Screening procedures which rely on nucleic acid
hybridization make it possible to isolate any gene sequence from
any organism, provided the appropriate probe is available.
Oligonucleotide probes, which correspond to a part of the neu
family of sequences provided herein and encoding a Neu protein
family member, can be synthesized chemically. This requires that
short, oligo-peptide stretches of the amino acid sequence be known.
The DNA sequence encoding the protein can be deduced from the
genetic code, however, the degeneracy of the code must be taken
into account. It is possible to perform a mixed addition reaction
when the sequence is degenerate. This includes a heterogeneous
mixture of denatured double-stranded DNA. For such screening,
hybridization is preferably performed on either single-stranded DNA
or denatured double-stranded DNA. Hybridization is particularly
useful in the detection of cDNA clones derived from sources where
an extremely low amount of mRNA sequences relating to the
polypeptide of interest are present. In other words, by using
stringent hybridization conditions directed to avoid non-specific
binding, it is possible, for example, to allow the autoradiographic
visualization of a specific cDNA clone by the hybridization of the
target DNA to that single probe in the mixture which is its
complete complement. (Wallace, et al., Nucl. Acid Res., 9:879,
1981). Alternatively, a subtractive library is useful for
elimination of non-specific cDNA clones.
[0079] Among the standard procedures for isolating cDNA sequences
of interest is the formation of plasmid- or phage-carrying cDNA
libraries which are derived from reverse transcription of mRNA
which is abundant in donor cells that have a high level of gene
expression. When used in combination with polymerase chain reaction
technology, even rare expression products can be cloned. In those
cases where significant portions of the amino acid sequence of the
polypeptide are known, the production of labeled single or
double-stranded DNA or RNA probe sequences duplicating a sequence
putatively present in the target cDNA can be employed in DNA/DNA
hybridization procedures which are carried out on cloned copies of
the cDNA which have been denatured into a single-stranded form
(Jay, et al., Nucl. Acid Res., 11:2325, 1983).
[0080] The nucleotide sequences of the present invention have a
myriad of applications. Representative uses of the nucleotide
sequences of the invention include the construction of cDNA and
oligonucleotide probes useful in Northern, Southern, and dot-blot
assays for identifying and quantifying the level of expression of
Neu family proteins in a cell. Lack of expression of a Neu protein
in tumors, diseased cells or tissues can indicate that measuring
the level of Neu expression can provide prognostic markers for
assessing the growth rate and invasiveness of a tumor.
[0081] In addition, considering the important role of Neu in
development and Ca.sup.2+ signaling, it is thought highly likely
that birth defects, degenerative, and psychiatric diseases can
result from expression of an abnormal Neu protein. In this case,
the Neu protein family can prove highly useful in prenatal
screening of mothers and/or for in utero testing of fetuses. Also,
early diagnosis of degenerative and neurological diseases can be
based on the analyses of changes in Neu expression and mutations in
the neu genes.
[0082] Similarly, the nucleotide sequences can be employed for the
construction of recombinant cell lines, ova, and transgenic embryos
and animals including dominant-negative and "knock-out" recombinant
cell lines in which the regulatory activity of Neu protein is
down-regulated or eliminated. Such cells can contain altered Neu
coding sequences that result in the expression of a Neu protein
that is not capable of enhancing, suppressing or activating
transcription of the target gene. The subject cell lines and
animals find use in screening for candidate therapeutic agents
capable of either substituting for a function performed by Neu or
correcting the cellular defect caused by a defective Neu.
[0083] The Neu family of proteins presents an attractive set of
diagnostic and therapeutic targets, considering the important
regulatory role this family of proteins plays in the development
function of adult organisms. This important role is reflected by
the effects one or defects in a mutant Neu protein can inflict upon
an organism. Moreover, with the advances in art of gene therapy
progresses, these defects can be correctable in utero or in early
post-natal life or alternatively through the use of compounds
identified in screening assays using Neu proteins. In addition, neu
polynucleotide molecules can be joined to reporter genes, such as
beta-galactosidase, luciferase, or green fluorescent proteins (GFP)
and inserted into the genome of a suitable host cell such as an
embryonic or tissue specific stem cell by, for example, homologous
recombination. Cells expressing neu can then be obtained by
subjecting the differentiating cells to cell sorting, leading to
the purification of a population of neu expressing cells. These
cells can be useful for studying specific activity of isolated cell
populations. Also, these cells can be used to study sensitivity to
growth factors or chemotherapeutic agents.
[0084] In yet another application of the nucleotide sequence
discovery, the technology can be useful in the construction of gene
transfer vectors (e.g., retroviral vectors, and the like). In these
vectors, the neu sequence is often inserted into the coding region
of the vector under the control of a promoter. neu gene therapy can
be used to correct neurological and movement diseases and cancer.
For these therapies, gene transfer vectors can either be injected
directly at the site of diseased cells, or the vectors can be used
to construct transformed host cells that are then injected at the
site of disease.
[0085] In one embodiment, a vector comprising a DNA molecule coding
a Neu protein is provided. Preferably, a DNA molecule coding a Neu
protein is inserted into a suitable expression vector, which is in
turn used to transfect or transform a suitable host cell. Exemplary
expression vectors for use in carrying out the present invention
include a promoter capable of directing the transcription of a
polynucleotide molecule of interest in a host cell. Representative
expression vectors include both plasmid and/or viral vector
sequences. Suitable vectors include retroviral vectors, vaccinia
viral vectors, CMV viral vectors, BLUESCRIPT (Stratagene, San
Diego, Calif.) vectors, baculovirus vectors, and the like. In
another embodiment, promoters capable of directing the
transcription of a cloned gene or cDNA can be inducible or
constitutive promoters and include viral and cellular promoters. In
particularly preferred embodiments, viral vectors are employed for
use in expressing Neu proteins in mammalian cells particularly if
neu is used for gene therapy.
[0086] In some embodiments, it can be preferable to use a
selectable marker to identify cells that contain the cloned DNA.
Selectable markers are generally introduced into the cells along
with the cloned DNA molecules and include genes that confer
resistance to drugs, such as neomycin, hygromycin, and
methotrexate. Selectable markers can also complement auxotrophies
in the host cell. Other selectable markers provide detectable
signals, such as beta-galactbsidase to identify cells containing
the cloned DNA molecules. Advantageously, the selectable markers
are amplifiable. Such amplifiable selectable markers can be used to
amplify the number of sequences integrated into the host
genome.
Antisense
[0087] Antisense neu nucleotide sequences can be used to block
expression of mutant neu expression in a variety of cell types.
Suitable antisense oligonucleotides are at least 11 nucleotides in
length and can include untranslated (upstream or intron) and
associated coding sequences. As will be evident to one skilled in
the art, the optimal length of an anti sense oligonucleotide
depends on the strength of the interaction between the antisense
oligonucleotide and the complementary mRNA, the temperature and
ionic environment in which translation takes place, the base
sequence of the antisense oligonucleotide, and the presence of
secondary and tertiary structure in the mRNA and/or in the
antisense oligonucleotide. Suitable target sequences for antisense
oligonucleotides include intron-exon junctions (to prevent proper
splicing), regions in which DNA/RNA hybrids will prevent transport
of mRNA from the nucleus to the cytoplasm, initiation factor
binding sites, ribosome binding sites, and sites that interfere
with ribosome progression.
[0088] Antisense oligonucleotides can be prepared, for example, by
the insertion of a DNA molecule containing the target DNA sequence
into a suitable expression vector such that the DNA molecule is
inserted downstream of a promoter in a reverse orientation as
compared to the gene itself. The expression vector can then be
transduced, transformed or transfected into a suitable cell
resulting in the expression of antisense oligonucleotides.
Alternatively, antisense oligonucleotides can be synthesized using
standard manual or automated synthesis techniques. Synthesized
oligonucleotides are introduced into suitable cells by a variety of
means including electroporation, calcium phosphate precipitation,
or microinjection. The selection of a suitable antisense
oligonucleotide administration method will be evident to one
skilled in the art. With respect to synthesized oligonucleotides,
the stability of antisense oligonucleotide-mRNA hybrids are
advantageously increased by the addition of stabilizing agents to
the oligonucleotide. Stabilizing agents include intercalating
agents that are covalently attached to either or both ends of the
oligonucleotide. In preferred embodiments, the oligonucleotides are
made resistant to nucleases by, for example, modifications to the
phosphodiester backbone by the introduction of phosphotriesters,
phosphonates, phosphorothioates, phosphoroselenoates,
phosphoramidates, phosphorodithioates, or morpholino rings.
Protein Production
[0089] As would be evident to one skilled in the art, the
polynucleotide molecules of the present invention can be expressed
in a variety of prokaryotic and eucaryotic organisms. For example,
the Neu family of proteins can be expressed in to, Saccharomyces
cerevisiae, filamentous fungi, and bacteria, such as E. coli to
produce Neu proteins. Similarly, one can express the protein of the
described invention in other host cells such as avian, insect, and
plant cells using regulatory sequences, vectors, and methods well
established in the literature.
[0090] Neu proteins produced according to the present invention can
be purified using a number of established methods such as affinity
chromatography using anti-Neu antibodies coupled to a solid
support. Fusion proteins of antigenic tag and Neu can be purified
using antibodies to the tag. Optionally, additional purification is
achieved using conventional purification means such as liquid
chromatography, gradient centrifugation, and gel electrophoresis,
among others. Methods of protein purification are known in the art
and can be applied to the purification of recombinant Neu described
herein.
Amino Acids
[0091] In one embodiment, the identification of mammalian Neu genes
is provided. Preferably, the mammalian Neu genes have highly
conserved sequences across the neuralized homology motif domains at
the amino acid level (Neu1, Neu 2, Neu3, and Neu 4). The following
Neu polypeptides or proteins have been identified: human neural
Neu1 protein of SEQ. ID. NO.:2, human muscle Neu1 protein SEQ. ID.
NO.:4; human Neu1 alternatively spliced form (h-neu1.DELTA.NHR1) of
SEQ. ID. NO.: 6; mouse neural Neu1 protein of SEQ. ID. NO.: 8;
mouse muscle Neu1 protein SEQ. ID. NO.: 10; mouse Neu1
alternatively spliced form (m-neu1.DELTA.NHR2A) of SEQ. ID. NO.:
12; mouse Neu1 alternatively spliced form (m-neu1.DELTA.NHR2B) of
SEQ. ID. NO.: 14; rat Neu 1 protein SEQ. ID. NO.: 16; rat Neu1
alternatively spliced form (r-neu1.DELTA.NHR2A) of SEQ. ID. NO.:
18; rat Neu1 alternatively spliced form (r-neu1.DELTA.NHR2B) of
SEQ. ID. NO.: 20; human Neu2 protein of SEQ. ID. NO.: 22; human
Neu2 alternatively spliced form (h-neu2.DELTA.NHR1) of SEQ. ID.
NO.: 24; human Neu2 alternatively spliced form (h-neu2.DELTA.NHR2)
of SEQ. ID. NO.: 26; rat Neu 2 protein SEQ. ID. NO.: 28; human Neu3
protein of SEQ. ID. NO.: 30; mouse Neu3 protein of SEQ. ID. NO.:
32; and human Neu4 protein (partial) of SEQ. ID. NO.: 34.
[0092] The described invention encompasses Neu variants that, for
example, are modified in a manner that results in Neu proteins
capable of translocating into the nucleus but unable to repress
transcription. Fragments of Neu proteins that are capable of
transcriptional repression but are incapable of translocating into
the nucleus are also encompassed by the present description.
Proteins retrieved from naturally occurring materials and closely
related, functionally similar proteins retrieved by antisera
specific to Neu, and recombinantly expressed proteins encoded by
genetic materials (DNA, RNA, cDNA) retrieved on the basis of their
similarity to the unique regions in the neu family of genes, are
also encompassed by the present description.
[0093] According to the present description, polynucleotide
molecules encoding Neu encompass those molecules that encode Neu
proteins or peptides that share identity with the sequences shown
in SEQ. ID. NOs.: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26,
28, 30, 32, and 34. Such molecules preferably share greater than
30% identity at the amino acid level with the disclosed sequences
in Neu. In preferred embodiments, the polynucleotide molecules can
share greater identity at the amino acid level across highly
conserved regions such as the neuralized homology repeat domains
and the RING-zinc finger domains.
[0094] It is contemplated that amino acid sequences substantially
the same as the sequences set forth in SEQ. ID. NOs.: 2, 4, 6, 8,
10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, and 34, are
encompassed by the described invention. A preferred embodiment
includes polypeptides having substantially the same sequence of
amino acids as the amino acid sequence set forth in SEQ ID NOs.: 2,
4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, and 34, or
functional fragments thereof, or amino acid sequences that are
substantially identical to SEQ ID NOs.: 2, 4, 6, 8, 10, 12, 14, 16,
18, 20, 22, 24, 26, 28, 30, 32, and 34. By "substantially the same"
or "substantially identical" is meant a polypeptide exhibiting at
least 80%, preferably 85%, more preferably 90%, and most preferably
95% homology to a reference amino acid sequence. For polypeptides,
the length of comparison sequences will generally be at least 16
amino acids, preferably at least 20 amino acids, more preferably at
least 25 amino acids, and most preferably 35 amino acids.
[0095] Homology is often measured using sequence analysis software
(e.g., Sequence Analysis Software Package of the Genetics Computer
Group, University of Wisconsin Biotechnology Center, 1710
University Avenue, Madison, Wis. 53705). Such software matches
similar sequences by assigning degrees of homology to various
substitutions, deletions, substitutions, and other
modifications.
[0096] The term "functional fragments" include those fragments of
SEQ ID NOs.: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28,
30, 32, and 34, or other Neu family members that retain the
function or activity of a Neu transcriptional regulator. One of
skill in the art can screen for the functionality of a fragment by
using the examples provided herein, where full-length Neu
transcriptional factors are described. It is also envisioned that
fragments of various Neu proteins that inhibit or promote
transcription can be identified in a similar manner. Neu
transcriptional activity can also be assayed by standard
transcription assays.
[0097] By "substantially identical" is also meant an amino acid
sequence which differs only by conservative amino acid
substitutions, for example, substitution of one amino acid for
another of the same class (e.g., valine for glycine, arginine for
lysine, etc.) or by one or more non-conservative substitutions,
deletions, or insertions located at positions of the amino acid
sequence which do not destroy the function of the protein assayed,
(e.g., as described herein). Preferably, such a sequence is at
least 85%, more preferably identical at the amino acid level to SEQ
ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32,
and 34.
[0098] By a "substantially pure polypeptide" is meant a Neu protein
that has been separated from components which naturally accompany
it. Typically, the polypeptide is substantially pure when it is at
least 60%, by weight, free from the proteins and naturally
occurring organic molecules with which it is typically associated.
Preferably, the preparation is at least 75%, more preferably at
least 90%, and most preferably at least 99%, by weight, Neu
protein. A substantially pure Neu polypeptide can be obtained, for
example, by extraction from a natural source; by expression of a
recombinant nucleic acid encoding a Neu polypeptide; or by
chemically synthesizing the protein. Purity can be measured by any
appropriate method, e.g., column chromatography, polyacrylamide gel
electrophoresis, or by HPLC analysis.
[0099] A protein is substantially free of naturally associated
components when it is separated from those contaminants that
accompany it in its natural state. Thus, a protein that is
chemically synthesized or produced in a cellular system different
from the cell from which it naturally originates will be
substantially free from its naturally associated components.
Accordingly, substantially pure polypeptides include those derived
from eukaryotic organisms but synthesized in E. coli or other
prokaryotes.
[0100] As would be evident to one skilled in the art, the
polynucleotide molecules of the present disclosure can be expressed
in a variety of prokaryotic and eucaryotic cells using regulatory
sequences, vectors, and methods well established in the
literature.
[0101] Neu proteins produced according to the present invention can
be purified using a number of established methods such as affinity
chromatography using anti-Neu antibodies coupled to a solid
support. Fusion proteins of antigenic tag and Neu can be purified
using antibodies to the tag. Optionally, additional purification is
achieved using conventional purification means such as liquid
chromatography, gradient centrifugation, and gel electrophoresis,
among others. Methods of protein purification are known in the art
and can be applied to the purification of recombinant Neu described
herein.
[0102] Construction of interspecies hybrid Neu proteins and hybrid
Neu proteins containing one or more domains from another Neu family
member are also contemplated. Such hybrid proteins facilitate
structure-function analyses. Similarly, hybrid proteins allow for
the alteration of Neu activity by increasing or decreasing the
transcriptional regulation of target genes. Hybrid proteins of the
present invention contain the replacement of one or more contiguous
amino acids of the native Neu with the analogous amino acid(s) of
Neu from another species or other protein of the Neu family. Such
interspecies or interfamily hybrid proteins include hybrids having
whole or partial domain replacements. Such hybrid proteins are
obtained using recombinant DNA techniques well known by one of
skill in the art. Briefly, DNA molecules encoding the hybrid Neu
proteins of interest are prepared using generally available methods
such as PCR mutagenesis, site-directed mutagenesis, and/or
restriction digestion and ligation. The hybrid DNA is then inserted
into expression vectors and introduced into suitable host
cells.
[0103] One embodiment of the present invention involves the
isolation of proteins that interact with Neu proteins and regulate
Neu protein function or are regulated by Neu. Neu proteins can be
used in immunoprecipitation to isolate interacting factors or used
for the screening of interactors using different methods of two
hybrid screening. Isolated interactors of Neu can be used to modify
Neu activity or Neu can be used to modify the activity of
interactors. Two hybrid screening has resulted in the isolation of
several types of interactors. Sequence analyses showed that all
interactors are novel proteins and contain RING-zinc finger domain
located in the C-terminus of the protein. NeuI-1 (4 clones) is a
novel splice variant (SEQ. ID. NOS.: 35, 36) of zinc finger protein
Miz1/PIASX.alpha./ARIP3 (GenBank accession numbers NM.sub.--008602;
AF077953; AF077954; AF044058). NeuI-2 (3 clones) is a fourth
homolog (SEQ. ID. NOS.: 37, 38; GenBank accession number AF277171;
AF302084) of zinc finger protein ZNF127 (GenBank accession numbers
U19106; U19107). NeuI-3 (9 clones) has highest homology to a human
hypothetical protein (GenBank accession number AK001459) and to a
Drosophila hypothetical protein (AAF56052.2) produced from CG4813
gene of a genomic scaffold (GenBank accession number AE003740)
(SEQ. ID. NOS.: 39, 40). NeuI-4 (12 clones) is the homolog of the
androgen receptor coactivator ARA54 (SEQ. ID. NO.: 32; GenBank
accession number AF060544) (SEQ. ID. NO.: 41, 42).
[0104] In still another embodiment, synthetic peptides,
recombinantly derived peptides, fusion proteins, chiral proteins
(stereochemical isomers, racemates, enantiomers, and D-isomers) and
the like are provided which include a portion of Neu or the entire
protein. The subject peptides have an amino acid sequence encoded
by a nucleic acid which hybridizes under stringent conditions with
an oligonucleotide of 15 or more contiguous nucleotides of SEQ. ID.
NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33.
Representative amino acid sequences of the subject peptides are
disclosed in SEQ. ID. NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22,
24, 26, 28, 30, 32, and 34. The subject peptides find a variety of
uses, including preparation of specific antibodies and preparation
of antagonists of Neu activity.
Antibodies
[0105] As noted above, the described teachings provide antibodies
that bind to Neu. The production of non-human antisera or
monoclonal antibodies (e.g., murine, lagomorph, porcine, equine) is
well known and can be accomplished by, for example, immunizing an
animal with Neu protein or peptides. Additionally, catalytic
antibodies to nuclear isoforms of the Neu family of proteins or Neu
protein metabolic intermediates that are transported into and out
of the nucleus can be generated. For the production of monoclonal
antibodies, antibody producing cells are obtained from immunized
animals, immortalized and screened, or screened first for the
production of the antibody that binds to the Neu protein or
peptides and then immortalized. It can be desirable to transfer the
antigen binding regions (e.g., F(ab')2 or hypervariable regions) of
non-human antibodies into the framework of a human antibody by
recombinant DNA techniques to produce a substantially human
molecule.
[0106] Following synthesis or expression and isolation or
purification of a Neu protein or a portion thereof, the isolated or
purified protein can be used to generate antibodies and tools for
identifying agents that interact with the Neu protein and fragments
of the Neu protein. Depending on the context, the term "antibodies"
can encompass polyclonal, monoclonal, chimeric, single chain, Fab
fragments and fragments produced by a Fab expression library.
Antibodies that recognize Neu proteins and fragments of Neu
proteins have many uses including, but not limited to,
biotechnological applications, therapeutic/prophylactic
applications, and diagnostic applications.
[0107] For the production of antibodies, various hosts including
goats, rabbits, rats, mice, etc. can be immunized by injection with
Neu proteins or any portion, fragment or oligopeptide that retains
immunogenic properties. Depending on the host species, various
adjuvants can be used to increase immunological response. Such
adjuvants include, but are not limited to, Freund's, mineral gels
such as aluminum hydroxide, and surface active substances such as
lysolecithin, pluronic polyols, polyanions, peptides, oil
emulsions, keyhole limpet hemocyanin, and dinitrophenol. BCG
(Bacillus Calmette-Guerin) and Corynebacterium parvum are also
potentially useful adjuvants.
[0108] Peptides used to induce specific antibodies can have an
amino acid sequence consisting of at least three amino acids, and
preferably at least 10 to 15 amino acids. Preferably, short
stretches of amino acids encoding fragments of Neu proteins are
fused with those of another protein such as keyhole limpet
hemocyanin such that an antibody is produced against the chimeric
molecule. While antibodies capable of specifically recognizing Neu
proteins can be generated by injecting synthetic 3-mer, 10-mer, and
15-mer peptides that correspond to a protein sequence of Neu
proteins into mice, a more diverse set of antibodies can be
generated by using recombinant Neu proteins, purified Neu proteins,
or fragments of Neu proteins.
[0109] To generate antibodies to Neu proteins and fragments of Neu
proteins, a substantially pure Neu protein or a fragment of Neu
protein is isolated from a transfected or transformed cell. The
concentration of the polypeptide in the final preparation is
adjusted, for example, by concentration on an Amicon filter device,
to the level of a few micrograms/ml. Monoclonal or polyclonal
antibody to the polypeptide of interest can then be prepared as
follows:
[0110] Monoclonal antibodies to Neu proteins or a fragment of Neu
proteins can be prepared using any technique that provides for the
production of antibody molecules by continuous cell lines in
culture. These include, but are not limited to, the hybridoma
technique originally described by Koehler and Milstein (Nature
256:495-497 (1975), the human B-cell hybridoma technique (Kosbor et
al. Immunol Today 4:72 (1983); Cote et al Proc Natl Acad Sci
80:2026-2030 (1983), and the EBV-hybridoma technique Cole et al.
Monoclonal Antibodies and Cancer Therapy, Alan R. Liss Inc, New
York N.Y., pp 77-96 (1985). In addition, techniques developed for
the production of "chimeric antibodies", the splicing of mouse
antibody genes to human antibody genes to obtain a molecule with
appropriate antigen specificity and biological activity can be
used. (Morrison et al. Proc Natl Acad Sci 81:6851-6855 (1984);
Neuberger et al. Nature 312:604-608(1984); Takeda et al. Nature
314:452-454(1985). Alternatively, techniques described for the
production of single chain antibodies (U.S. Pat. No. 4,946,778) can
be adapted to produce Neu protein-specific single chain antibodies.
Antibodies can also be produced by inducing in vivo production in
the lymphocyte population or by screening recombinant
immunoglobulin libraries or panels of highly specific binding
reagents as disclosed in Orlandi et al., Proc Natl Acad Sci 86:
3833-3837 (1989), and Winter G. and Milstein C; Nature 349:293-299
(1991).
[0111] Antibody fragments that contain specific binding sites for
Neu proteins can also be generated. For example, such fragments
include, but are not limited to, the F(ab').sub.2 fragments that
can be produced by pepsin digestion of the antibody molecule and
the Fab fragments that can be generated by reducing the disulfide
bridges of the F(ab').sub.2 fragments. Alternatively, Fab
expression libraries can be constructed to allow rapid and easy
identification of monoclonal Fab fragments with the desired
specificity. (Huse W. D. et al. Science 256:1275-1281 (1989)).
[0112] By one approach, monoclonal antibodies to Neu proteins or
fragments thereof are made as follows. Briefly, a mouse is
repetitively inoculated with a few micrograms of the selected
protein or peptides derived therefrom over a period of a few weeks.
The mouse is then sacrificed, and the antibody producing cells of
the spleen isolated. The spleen cells are fused in the presence of
polyethylene glycol with mouse myeloma cells, and the excess
unfused cells destroyed by growth of the system on selective media
comprising aminopterin (HAT media). The successfully fused cells
are diluted and aliquots of the dilution placed in wells of a
microtiter plate where growth of the culture is continued.
Antibody-producing clones are identified by detection of antibody
in the supernatant fluid of the wells by immunoassay procedures,
such as ELISA, as originally described by Engvall, E., Meth.
Enzymol. 70:419 (1980), and derivative methods thereof. Selected
positive clones can be expanded and their monoclonal antibody
product harvested for use. Detailed procedures for monoclonal
antibody production are described in Davis, L. et al. Basic Methods
in Molecular Biology Elsevier, New York. Section 21-2.
[0113] Polyclonal antiserum containing antibodies to heterogenous
epitopes of a single protein can be prepared by immunizing suitable
animals with the expressed protein or peptides derived therefrom
described above, which can be unmodified or modified to enhance
immunogenicity. Effective polyclonal antibody production is
affected by many factors related both to the antigen and the host
species. For example, small molecules tend to be less immunogenic
than others and can require the use of carriers and adjuvant. Also,
host animals vary in response to site of inoculations and dose,
with both inadequate or excessive doses of antigen resulting in low
titer antisera. Small doses (ng level) of antigen administered at
multiple intradermal sites appears to be most reliable. An
effective immunization protocol for rabbits can be found in
Vaitukaitis, J. et al. J. Clin. Endocrinol. Metab. 33:988-991
(1971).
[0114] Booster injections can be given at regular intervals, and
antiserum harvested when antibody titer thereof, as determined
semi-quantitatively, for example, by double immunodiffusion in agar
against known concentrations of the antigen, begins to fall. See,
for example, Ouchterlony, O. et al., Chap. 19 in: Handbook of
Experimental Immunology D. Wier (ed) Blackwell (1973). Plateau
concentration of antibody is usually in the range of 0.1 to 0.2
mg/ml of serum (about 12 .mu.M). Affinity of the antisera for the
antigen is determined by preparing competitive binding curves, as
described, for example, by Fisher, D., Chap. 42 in: Manual of
Clinical Immunology, 2d Ed. (Rose and Friedman, Eds.) Amer. Soc.
For Microbiol., Washington, D.C. (1980). Antibody preparations
prepared according to either protocol are useful in quantitative
immunoassays that determine concentrations of antigen-bearing
substances in biological samples; they are also used
semi-quantitatively or qualitatively (e.g., in diagnostic
embodiments that identify the presence of a Neu protein in
biological samples). It is also contemplated that various methods
of molecular modeling and rational drug design can be applied to
identify additional Neu protein family members, compounds that
resemble a Neu protein or fragment or derivative thereof, and
molecules that interact with Neu proteins and, thereby modulate
their function.
Additional Applications
[0115] The Neu family of proteins presents an attractive set of
diagnostic and therapeutic targets, considering the important
regulatory role this family of proteins plays in the development
function of adult organisms. This important role is reflected by
the effects one or more defects in a mutant Neu protein can inflict
upon an organism. Moreover, with the advances in the art of gene
therapy, these defects can be corrected in utero or in early
post-natal life or alternatively through the use of compounds
identified in screening assays using Neu proteins.
[0116] In some instances, cancer cells, or diseased cells or
tissue, can contain a non-functional Neu protein or can contain no
Neu protein due to a genetic mutation or somatic mutations such
that these cells fail to stop proliferating and differentiate. For
cancers of this type, the cancer cells can be treated in a manner
to cause the over-expression of wild-type Neu protein to force
differentiation and cease proliferation of the cancer cells.
Accordingly, a method of treating cancer is similarly provided.
[0117] It is also contemplated that because neu family genes
control cell proliferation and Ca.sup.2+ signaling induced
transcriptional processes, that manipulating Neu expression and
function may be useful in controlling a variety of diseases, a few
examples of which include depression, pain, anxiety,
neurodegenerative diseases, and cancer.
[0118] The practice of the described invention is illustrated in
the following non-limiting examples. The examples are provided
below are not intended to limit the invention in any way.
EXAMPLE 1
Characterization of neu1 Transcripts
[0119] A mouse cDNA library of postnatal day (P) 1 brain
(Stratagene, San Diego, Calif.) was screened with a mouse 0.7 kb
EST (GenBank #AA5 18339) cDNA clone corresponding to a region
ranging from about the translation initiation codon to the end of
the first neuralized homology repeat domain (NHR1) of h-neu1
(Nakamura et al., 1998).
[0120] Sequence analyses, the results of which are shown in FIG. 1,
revealed that the isolated cDNA clones differed in their 5' regions
encoding muscle- and brain-specific m-Neu1 proteins with different
N-termini (FIG. 1). In the figure, "A" shows an amino acid sequence
comparison of Drosophila, human, rat and mouse neu1 proteins.
Various domain regions of the proteins are illustrated. The regions
Brain-N, relating to the neural-specific N-terminal region, and
Muscle-N, the muscle-specific N-terminal of mammalian neu proteins,
are boxed. The NHR1 and NHR2 regions, neuralized homology repeat
domains 1 and 2; RZD, RING zinc finger domain are underlined. NLS1
and NLS2, nuclear localization signal sequences, are also
shown.
[0121] RT-PCR analyses of RNA from mouse and rat brain and skeletal
muscle resulted in identification of 6 different Neu1 transcripts
in both species (FIG. 1): 1) brain- and muscle-specific transcripts
encoding Neu1 with the intact NHR2 domain (574 and 557 amino acids,
respectively); 2) brain- and muscle-specific transcripts encoding
Neu1 protein isoforms that lack the region between NHR1 and NHR2,
the entire NHR2 region and different parts of the linker region
preceding the RZD (Neu1-.DELTA.NHR2A, 342 and 325 amino acids and
Neu1-.DELTA.NHR2B, 291 and 274 amino acids).
[0122] Turning again to FIG. 1, the region between asterisks is
absent in the splice isoform neu1-.DELTA.NHR2A; the region between
the open circles is absent in the splice isoform neu1-.DELTA.NHR2B;
the pKB consensus sequence between NHR1 and NHR2; LRS, two putative
leucine rich sequences in the end of both the NHR; and SP, the
serine and proline rich repeats between the NHR2 and RZD are boxed.
"B" is a schematic representation of the domain structure of
different mouse and rat Neu1 isoforms. Structures of the full
length Neu1 protein (neu, 574 amino acids) and of two Neu1 isoforms
(neu-.DELTA.NHR2A, 342 amino acids, neu-.DELTA.NHR2B, 291 amino
acids) are shown. Lines indicate to the alternative splicing
resulting in the cDNAs encoding neu-.DELTA.NHR2A and
neu-.DELTA.NHR2B isoforms. The numbers below the neu-.DELTA.NHR2A
and neu-.DELTA.NHR2B correspond to the amino acids of the full
length Neu1 protein.
[0123] Sequence analyses revealed that alternative splicing in the
NHR2 and RZD linker region occurs in-frame and does not affect the
intactness of the RING zinc finger structure. Accordingly, both
m-neu1 and r-neu1 genes encode protein isoforms with one or two
NHRs followed by the C3HC4-type RZD in the C-terminus. To date no
proteins other than Neu1 proteins have been identified that contain
NHR-like domains and the function of the NHR-like domains has not
been previously identified. The RING zinc finger motifs are present
in many regulatory proteins and have been shown to mediate
protein-protein interactions. Saurin et al., Trends Biochem Sci,
21:208-214 (1996).
[0124] Like h-Neu, m-Neu1 and r-Neu do not have sequences that are
similar to the Lys-rich nuclear localization signal (NLS) in d-Neu.
However, two smaller clusters of Arg and Lys rich amino acids
(HKAVKAR (SEQ ID NO: 43) at 80-85 and RLKITKK (SEQ ID NO: 44) at
107-113) are present in the NHR1 of mouse, rat, and human Neu1 that
resemble the phosphorylation consensus sequence of NLS, m-Neu1,
r-Neu, and h-Neu proteins, which are rich in Ser and Thr residues.
The presence of these regions suggests that Neu1 is regulated by
phosphorylation. The region between NHR1 and NHR2 contains also a
putative protein kinase B/AKT phosphorylation site RPRSFT (SEQ ID
NO: 45) which is similar to the respective consensus sequence
RXRXXS/T (SEQ ID NO: 46). Datta, et al., Genes and Dev, 13:2905-27
(1999).
[0125] The region between NHR2 and RZD of m-Neu1, r-Neu, and h-Neu
contains two imperfect repeats of Pro, Ser, and Thr residues with
the consensus sequence S/TXPXSPXSXPXSPXXXGXXX(X)SD (SEQ ID NO: 47)
where X denotes any amino acid. It is interesting to note that this
SP repeat of mammalian Neu1 proteins is not similar to any known
protein motifs.
EXAMPLE 2
Developmentally Regulated Expression of neu1 mRNA in the Mouse and
Rat Skeletal Muscle and Brain
[0126] An RNase protection analyses (RPA) was used to determine the
levels of m-neu1 and r-neu1 mRNAs in the developing and adult brain
and non-neural tissues (FIG. 2).
[0127] Total RNA was isolated from the mouse and rat brain regions,
which are indicated (A) and (B) respectively in FIG. 2, and
non-neural tissues. Levels of neu1 transcripts were analyzed by
RNase protection assays. The cRNA probes used for detection of
m-neu1 transcripts were complementary to a region encoding the
muscle-specific (FIG. 2, left panel) or the brain-specific (FIG. 2,
right panel) N-terminus and first half NHR1 of m-neu1. For r-neu1,
the cRNA probe was complementary to a region encoding the second
half of NHR2 up to the stop codon. Specific protected fragments are
indicated on the right of each panel.
[0128] Bottom panels of FIG. 2 show the levels of GAPDH mRNA in the
RNA samples. The nomenclature used to identify each sample is as
follows: neu-M, indicates a muscle-specific m-neu1 transcript;
neuB, indicates a brain-specific m-neu1 transcript; neu-B/M
indicates the total pool of muscle- and brain-specific r-neu1
transcripts. Further, E, denotes "Embryonic day"; P, denotes
"postnatal day"; ad, denotes "adult"; th, denotes "thymus"; he,
denotes "heart"; lu, denotes "lung"; sp, denotes "spleen"; ki,
denotes "kidney"; li, denotes "liver"; mu, denotes "skeletal
muscle"; gu, denotes "gut"; plac, denotes "placenta"; ctx, denotes
"cerebral cortex"; cbl, denotes "cerebellum"; hc, denotes
"hippocampus"; str, denotes "striatum"; mid, denotes "ventral
midbrain"; hth, denotes "hypothalamus"; col, denotes "colliculi";
thal, denotes "thalamus"; pons, denotes "pons"; olf; denotes
"olfactory bulb"; med, denotes "medulla"; pit, denotes "pituitary";
and tRNA, denotes "yeast tRNA", which was used as a negative
control.
[0129] The overall highest expression levels of neu1 mRNA were seen
in the adult skeletal muscle and brain. In the skeletal muscle,
neu1 mRNA levels were undetectable during embryonic development,
low at birth and upregulated during postnatal development reaching
the highest levels in the adult. Other non-neural tissues (heart,
kidney, liver, lung, thymus, and spleen) except for the adult heart
and testis did not express neu1 transcripts or the levels were
below the detection limit of the RPA. In the brain, low levels of
neu1 mRNA were detected at embryonic day (E) 13 and the expression
increased progressively reaching the highest levels in the adult.
neu1 expression levels were high in the cerebral cortex,
hippocampus, and striatum and substantially lower in the olfactory
system, thalamus/hypothalamus, midbrain, cerebellum, pons, and
medulla.
[0130] neu1 expression levels were specifically analyzed during
postnatal development of cerebral cortex and cerebellum. In both of
these brain regions, low levels of neu1 mRNA were observed in
postnatal day (P) 1 whereas by two weeks after birth the expression
levels increased significantly reaching the peak levels in the
adult animal. High levels of neu1 mRNA expression were also
detected in the adult dorsal root ganglia and moderate levels in
the adult spinal cord. In all the tissues predominant neu1
transcripts contained intact NHR2 domain, whereas the levels the
neu1 mRNAs lacking the NHR2 (neu1-.DELTA.NHR1A and
neu1-.DELTA.NHR1B) were below 5% of all m-neu1 transcripts.
Presented data show that neu1 is highly expressed during mouse and
rat postnatal development and that the expression is confined to
skeletal muscle and the nervous system.
EXAMPLE 3
Neuronal Expression and Dendritic Localization of Mammalian
Neuralized mRNA
[0131] In situ hybridization analysis was used to study the
cellular localization of neu1 mRNA expression in the embryonic and
adult mouse brain. The results are presented in FIG. 3.
[0132] Shown are dark-field emulsion autoradiographs obtained after
hybridization of coronal sections of mouse brain with the
[.sup.35S]-labeled m-neu1 cRNA probe corresponding to the first
neuralized homology region (NHR1) of neu1. m-neu1 mRNA-specific
labeling is shown at embryonic day 17 (E17, upper panel), at
postnatal day 7 (P7, middle panel) and adult (AD, lower panel) in
different brain structures. Exposure time was 3 weeks for adult and
P7 sections and 6 weeks for E17 sections. As shown in the figure,
BG, denotes "basal ganglia"; Cx, denotes "cerebral cortex"; Th,
denotes "thalamus"; PTec, denotes "pretectum"; SC, denotes
"superior colliculi"; IC, denotes "inferior colliculi"; Cb, denotes
"cerebellum"; Me, denotes "medulla"; Pit, denotes "pituitary"; P,
denotes "pons"; Hc, denotes "hippocampus"; Am, denotes "amygdala";
and Pn, denotes "Pontine nuclei".
[0133] Results of these analyses supported the conclusions drawn
from the RPA studies discussed in Example 2. Neuralized1
mRNA-specific labeling was not detected at E13 and E15 brain,
because of very low expression levels. This result was supported by
the related data produced from the RPA. At E17, all brain regions,
except for the cerebral cortex, hippocampus, and cerebellum, were
found to express low levels of m-neu1 mRNA. In the brain, m-neu1
expression was confined to the regions containing postmitotic
neurons and was not present in ventricular and subventricular zones
that contain proliferating neural stem and progenitor cells.
[0134] At P7, the levels of neu1 mRNA increased significantly in
the basal ganglia, amygdala, hypothalamus, and hippocampus. Low
levels were present in the cerebral cortex and brainstem. In the
adult, widespread expression of m-neu1 mRNA was observed throughout
the brain particularly in the cerebral cortex, hippocampal
formation, the basal ganglia, amygdaloid, hypothalamus, and pontine
nuclei and in the cerebellum, while lower levels were seen in the
mesencephalon and medulla oblangata. m-neu1 mRNA-specific signal
was not detected in most of the thalamic nuclei.
[0135] A more detailed analysis of neu1 expression was carried out
in rat brain and the data is shown in Table 2 and in FIGS. 4, 5,
and 6.
[0136] FIG. 4 shows the results of an in situ hybridization
analysis of neu1 mRNA expression in adult rat brain. The coronal
sections (A-H) correspond to the levels from 5 Bregma in the atlas
of Paxinos and Watson (Paxinus and Watson, 1986) as indicated by
numbers in the left part of the bottom of each autoradiograph. CTX,
denotes "cerebral cortex"; CPu, denotes "caudate putamen"; Pir,
denotes "piriform cortex"; LS, denotes "lateral septum"; HDB,
denotes "nucleus of the horizontal limb of the diagonal band of
Broca"; VDB, denotes "nucleus of the vertical limb of the diagonal
band of Broca"; GP, denotes "globus pallidus", BSTM and BSTL,
denotes "bed nucleus of the stria terminalis, medial and lateral
division respectively"; MHb, denotes "medial habenular nucleus";
Rt, denotes "reticular thalamic nucleus"; AH, denotes "anterior
hypothalamic area"; Zi, denotes "zona incerta"; Am, denotes
"amygdaloid nuclei"; VMH, denotes "ventromedial hypothalamic
nucleus"; DMD, denotes "dorsomedial hypothalamic nucleus"; DG,
denotes "dentate gyrus"; CA1 and CA3, denotes "pyramidal layers of
the hippocampus"; Ent, denotes "entorhinal cortex"; PAG, denotes
"periaqueductal gray"; Sc, denotes "superior colliculus"; S,
denotes "subiculum"; Pn, denotes "pontine nuclei"; GL, denotes
"granular layer of cerebellum"; PL, denotes "Purkinje cell layer of
cerebellum"; ECu, denotes "external cuneate nucleus"; Ve, denotes
"vestibular nuclei"; Gi, denotes "gigantocellular reticular
nucleus"; Sp5, denotes "spinal trigeminal nucleus": PCRt and IRt,
denote "parvicellular reticular nuclei" and "intermedial reticular
nuclei", respectively; LPGi, denotes "lateral paragigantocellular
nucleus"; LRt, denotes "lateral reticular nucleus"; and Sol,
denotes "solitary nucleus". The scale bar corresponds to 5 mm in
length.
[0137] FIG. 5 shows expression of neu1 mRNA in an adult rat nervous
system. Dark-field in situ hybridization autoradiographs showing
labelling in A, the cerebral cortex; B, CA1-CA3, hippocampal
subfields, and in the dentate gyrus (DG) including the strata
molecular (Mol), lacunosum molecular (Lmol) and radiatum (Rad); C,
substantia nigra compact part (SNc) and reticular part (SNr); D,
medial habenular nucleus (MHb); E, locus coeruleus (LC); F, in the
molecular (Mol) and granular (Gr) layer of cerebellar cortex; G, in
the layers of the retina: ganglion cell layer (GCL), inner
plexiform layer (IPL), inner nuclear layer (INL), outer plexiform
layer (OPL), outer nuclear layer (ONL), pigmental epitelial (PE);
H, dorsal root ganglia (DRG). Roman numbers in A indicate the
various layers of cerebral cortex; CC, corpus callosum; Hil, hilus;
WM, white matter. The scale bars correspond lengths of 200 .mu.m in
A and 400 .mu.m in B-H.
[0138] FIG. 6 shows cellular localization of neu1 mRNA in an adult
rat nervous system. Bright-field in situ hybridization
microphotographs showing labelled cells in A, in the layer II of
the cerebral cortex at the level of the somatosensory cortex (CTX);
B, dentate gyrus (DG) of hippocampus; C, CA3 layer of the
hippocampus; D, polymorph layer of dentate gyrus (PoDG); E, lateral
septum (LS); F, substantia nigra compact part (SNc); G, globus
pallidus (GP); H and I, spinal cord at L3 level, respectively in
the layers 9 and 2-3 (Sc); J, dorsal root ganglia (DRG); K, glia
limitans (GL); L, pineal gland (Pi). Note in G, the cells in the
globus pallidus (GP) do not express neu1 mRNA. Mol, stratum
moleculare; Hil, hilus of the hippocampus; Rad, stratum radiatum;
Or, stratum oriens. The scale bar corresponds to 25 .mu.m in
length.
[0139] Overall, although the neu1-specific labeling was distributed
broadly throughout the brain, more predominant expression was
confined to forebrain structures and the levels were lower in
caudal regions of the brain, with the exception of some nuclei
(Table 2). In the olfactory system the anterior olfactory nucleus
showed neu1-specific signal. In the cerebral cortex neu1 mRNA
expression levels were particularly high (FIG. 4A-F, 5A, 6A). In
the cerebral cortex, highest levels of neu1 mRNA expression were
found in layer I-III, whereas expression gradually decreases from
layer IV to layer VI. Dense labeling was seen in layers I-III, and
in scattered neurons of layer V, moderate levels in layers V and
VI, and low levels in layer IV. (FIG. 5A, 6A) Interestingly,
labeling in layer I did not cover diffuse cell bodies, suggesting
dendritic localization of neu1 mRNA. Piriform and entorhinal
corticies were also labeled with neu1-specific signal.
[0140] In the hippocampus, neurons of the granular layer of dentate
gyrus and the pyramidal layers CA1-CA3 showed moderate-high
labeling (FIG. 4C-E, 5B, 6B-D). Neurons of the hilus and subiculum
expressed low levels of neu1 mRNA. The most interesting observation
regarding neu1 mRNA localization in the adult brain was the clear
dendritic localization of the transcripts in the hippocampus. The
neu1-specific labeling over the molecular layer of the dentate
granule cells was uniformly distributed (FIG. 4C-E). Examination of
emulsion-dipped sections showed that this labeling did not cover
cell bodies and extended up to the hippocampal fissure, showing
that neu1 mRNA is localized throughout the entire dendritic tree of
dentate granule cell layer (FIG. 5B, 6B). The strata oriens and
radiatum of CA1-CA3 of hippocampus, corresponding to the dendrites
of these regions, were also labeled by neu1 cRNA but the levels
were significantly lower than in the molecular layer of the dentate
gyrus (FIG. 4C-E, 5B, 6C).
[0141] In the basal ganglia, r-neu1 cRNA was detected in caudate
putamen and accumbens but not in the globus pallidus (FIG. 4A, B,
6G). Moderate to high levels of neu1 mRNA were seen in all the
amygdaloid nuclei (FIG. 4C-D), the interstitial nucleus of the
posterior limb of anterior commissure and the bed of stria
terminalis of extended amygdala (FIG. 4B). In the septum, high
levels of labeling were seen in the cells of lateral septum (FIG.
4A; 6E), and very low levels in the vertical and horizontal limb of
the diagonal band of Broca. In the hypothalamus, the supraoptic,
suprachiasmatic, supramammilary, and ventromedial nuclei expressed
moderate levels, and several other nuclei expressed low levels of
neu1 mRNA (FIG. 4B-E). In the thalamus, the medial habenula
expressed high levels of neu1 mRNA, while moderate levels were
observed in the reticular thalamic nucleus and low levels in a few
other nuclei of the thalamus, such as the zona incerta, lateral
habenula, mediodorsal thalamic nuclei, laterodorsal thalamic
nuclei, paraventricular thalamic nucleus, rheuniens, rhomboid
thalamic nuclei, and medial pretectal nucleus (Table 1, FIG. 4C-D,
5D). In the mesencephalon, the neurons of the substantia nigra,
colliculi superior, ventral tegmental area, periaqueductal gray,
interpeduncular nucleus, oculomotor nucleus, and Raphe nucleus
showed low levels of neu1-specific signal (FIG. 4F, 5C, 6F). The
granule cell layer of the cerebellum was strongly labeled, but the
Purkinje cells and deep cerebellar nuclei were not (FIG. 4G, H,
5F). Within myelenchepalon, the locus coeruleus, and the pontine,
trapezoid body, facialis, pontine reticular, and
rostroventrolateral reticular nuclei were labeled with moderate
density with r-neu1 cRNA (Table 1, FIG. 4F, 5E). Beyond this, there
was low-density labeling in the trigeminal, facial, solitary, and
hypoglossal nuclei (FIG. 4G, H). In addition, neu1 cRNA labeling
was evident at low levels or in association with fewer cells in
several other nuclei, such as the vestibular, cochlear,
paragigantocellular, and abducens nuclei (Table 1).
[0142] In the retina, labeling of neu1 mRNA was strong in the inner
nuclear layer, with the highest intensity in the outer border.
Diffuse labeling was also seen in the ganglion cell layer and both
in the inner and outer plexiform layers (FIG. 5G). Cells in the
pineal gland expressed high levels of neu1 mRNA (FIG. 6L). Finally,
labeling was relatively low in the cells of the spinal cord (FIG.
6H, I), in contrast to the neurons of dorsal root ganglia, which
expressed high levels of neu1 mRNA (FIG. 5H, 6J).
[0143] At the cellular level, m-neu1 mRNA expression was
predominant in cells displaying neuronal profile (large cells with
weakly stained nuclei) and not in the cells with glial profile
(small cells with strongly stained nuclei). In addition, neu1 mRNA
expression was not detected in the white matter, where neuronal
cells are absent. One of the few exceptions of non-neuronal neu1
mRNA expression was glia limitans that displayed dense labeling
(FIG. 6K).
[0144] Using in situ hybridization in combination with
immunocytochemistry for NeuN, a Neuron-specific protein, we found
that clusters of silver grains, reflecting the presence of neu1
mRNA, were located over the perikarya of NeuN-positive cells in all
the brain regions examined, such as cerebral cortex, hippocampal
formation, striatum, hypothalamus nuclei, amygdaloid complex, and
cerebellum. FIG. 7 shows the results of neuronal expression studies
of neu1 mRNA levels in adult rat brain In the figure, neu1 mRNA was
visualized as autoradiographic grains whereas NeuN was shown as
peroxidase staining resulting in a yellow color. The two labelings
co-localized with all the cells. As shown in the figure: A, layer
II of cerebral cortex (CTX); B, CA1 layer of the hippocampus; C,
polymorph layer of the dentate gyrus (PoDG) and CA3 layer of the
hippocampus; D, caudate putamen (CPu); E, reticular thalamic
nucleus (Rt); and F, ventromedial hypothalamic nucleus (VMH), were
studied Note that neurons (NeuN-positive cells) of the ventral
posterolateral thalamic nucleus (VPL) in E do not express neu1
mRNA.
[0145] By evaluation the proportion of cells positive for both neu1
mRNA and NeuN out of the total number of NeuN-containing neurons,
it was found that all the neurons in the layers II and III of the
cerebral cortex expressed neu1 mRNA. In the layer IV eighty percent
and layers V-VI about ninety percent of neurons expressed neu1
mRNA. In several other brain regions examined, such as hippocampal
formation, striatum, reticular thalamic nucleus, hypothalamic
nuclei, showing that the majority of the neuronal cells express
neu1 mRNA. In contrast, neurons in the globus pallidus and in
several thalamic nuclei, such as ventral posterolateral and
posteromedial nuclei, did not express neu1 mRNA (FIG. 7).
[0146] Neuronal activity has been shown to regulate the dendritic
localization of the immediate-early gene arc mRNA. Steward et al.,
Neuron, 21:741-51 (1998); Guzovsky et al., Nat Neurosci,
2:1120-1124 (1999). To examine if neu1 mRNA dendritic localization
is regulated by neuronal activity, the effect of kainate-induced
seizures on the expression and localization of neu1 mRNA was
studied (FIG. 8). At 24 hours after the injection of kainate, neu1
mRNA levels decreased in all the regions of the hippocampus. The
levels of neu1 mRNA are significantly downregulated in the granular
and molecular layers of dentate gyrus of adult rat brain at 24
hours after the treatment with kainic acid (B and B1), as compared
to control brains (A and A1). Regions analyzed are denoted as
follows: CTX, cerebral cortex; DG, dentate gyrus; Am, amygdala;
Pir, piriform cortex; VMH, ventromedial hypothalamic nucleus; and
Mol, for the molecular layer of the hippocampus. Particularly
pronounced was the reduction of labeling in the molecular layer of
the dentate gyrus. Similarly, but less consistently, a
down-regulation of neu1 mRNA was observed in the cerebral
cortex.
EXAMPLE 4
Neu1 Protein Isoforms Exhibit Transcriptional Repressor
Activities
[0147] The ability of m-Neu1 to act as a transcriptional regulator
was studied using a chloramphenicol acetyl-transferase (CAT) assay.
Several transcriptional regulators, such as the polycomb
group-related transcriptional regulator MEL18, the MDM2
proto-oncogene, breast and ovarian cancer susceptibility gene BRCA1
and MAT1, a subunit of TFIIH basal complex factor contain RING
finger motifs. Kanno et al., EMBO J, 14:5672-5678 (1995);
Leveillard and Wasylyk, J Biol Chem, 272:30651-30661 (1997); Pao et
al., Proc Natl Acad Sci USA, 97:1020-1025 (2000); and Fesquet et
al., Oncogene, 15:1303-1307 (1997). Furthermore, the earlier
studies proposed d-Neu to function as a DNA-binding transcription
factor. Boulianne et al., EMBO J, 10:2975-2983 (1991) and Price et
al., EMBO J, 12:2411-2418 (1993).
[0148] The effect of m-Neu1 on transcriptional activity was studied
using a chloramphenicol acetyl-transferase (CAT) assay and the
following promoters: 1) TATA-box containing: 1.0 kb BDNF-I-CAT, 0.3
kb BDNF-II-CAT, 0,7 kb BDNF-IV CAT, 0.4 kb NF-L, 1.0 kb GAP-43, 2)
TATA-less promoters with putative initiators (Inr): 0.4 kb
BDNF-III-CAT, 0.4 kb LNGFR-CAT, and 0.3 kb ME1-CAT. Timmusk et al.,
Neuron, 10:475-489 (1993); Reeben et al., J Neurosci Res,
40:177-188 (1995); Chiaramello et al., J Biol Chem, 271:22035-22043
(1996); Metsis et al., Gene, 121:247-254 (1992); and Shain et al.,
Nucleic Acids Res, 23:1696-1703 (1995). Data from the study is
shown in FIG. 9.
[0149] FIG. 9 show that Neu1 represses transcription from various
promoters in transient expression assays. The data for this figure
was gathered from Neuro2A cells that were co-transfected with 0.5
.mu.g of various reporter plasmids containing different promoters
driving the epression of CAT and pRcCMV expression plasmid (1.0
.mu.g) without any cDNA sequence (control) or containing m-neu1
cDNAs encoding the full length protein (neu-FL) or the
m-Neu-1-.DELTA.NHR2A (neu.DELTA.NHR2A) isoform lacking the region
between NHR1 and RZD, using FuGENE-6 transfection system. The
pON260 expression plasmid (0.1 .mu.g) encoding .beta.-galactosidase
was included in the transfections to normalize the transfection
efficiencies. The CAT activity is defined as 100 for each reporter
when cotransfected with the pRcCMV parental expression plasmid.
When cotransfected with expression plasmids containing m-neu1
cDNAs, the CAT activities are expressed relative to the value
obtained by cotransfection of each reporter plasmid and the
parental pRcCMV expression plasmid. The data shown are
representative of at least three independent experiments. Error
bars represent the S.E. BDNF-I, 1.0 kb BDNF promoter I, BDNF II,
0.3 kb BDNF promoter II; BDNF III, 0.4 kb BDNF promoter III; BDNF
IV, 0.7 kb BDNF promoter IV; NF-L, 0.4 kb NF-L promoter; GAP-43,
1.0 kb GAP-43-promoter, LNGFR, 0.4 kb LNGFR promoter; ME1, 0.3 kb
ME1 promoter.
[0150] The full-length and the isoform of m-Neu1-.DELTA.NHR2A
significantly reduced the activity of all of these promoters in
Neuro-2A cells (FIG. 9). CAT assays performed in other cell types
(mouse teratocarcinoma PCC7, rat astrocytoma C6, human breast
ductal carcinoma BT-549, human primary osteogenic sarcoma Saos2,
human cervix carcinoma C-33A) or using a different reporter
(RAR.beta. promoter driven lac Z) gave similar results (data not
shown), suggesting that the transcriptional repressor activity of
m-Neu1 does not depend on the reporter and cellular context.
[0151] Following the identification of m-Neu1's activity as a
transcriptional regulator, identification of the repression domains
of m-Neu1 was performed. Expression plasmids were generated
encoding full-length m-Neu1 or individual domains of m-Neu1 fused
to Gal4 DNA-binding domain and tested their activities by CAT
assays. The data is presented in FIG. 10.
[0152] FIG. 10 shows that neuralized homology repeat domains of
Neu1 mediate the transcriptional repression when fused to the DNA
binding domain of Gal4. Neuro2A cells were cotransfected with
Gal4TK-CAT reporter plasmid containing five Gal4 binding sites in
front of TK promoter driving the expression of CAT (0.5 .mu.g) and
pBIND expression plasmid (1.0 .mu.g) without any cDNA sequence (G4)
or containing different regions of m-neu1 cDNA fused in-frame to
Gal4 DNA-binding domain using FuGENE-6 transfection system. The
designations of the constructs are shown on the left of FIG. 10.
The CAT activities are expressed relative to the value obtained by
cotransfection of the reporter plasmid and the parental pRcCMV
expression plasmid which was set 100. The data shown are
representative of at least three independent experiments. Error
bars represent the S.E.
[0153] The co-transfection of full-length m-Neu1 fused to Gal4
(Gal4-m-Neu1) with the Gal4-TK-CAT resulted in a
concentration-dependent repression of the CAT activity (FIG. 10).
m-Neu1 isoforms that lack the NHR2 and the region preceding the RZD
also displayed transcriptional repressor activities when fused to
Gal4 DBD (FIG. 10). Extensive deletion analyses showed that NHR1
and NHR2 domains possess transcriptional repressor activity,
whereas neural and muscle-specific N-termini (amino acids 1-60),
RZD (amino acids 509-574) and the serine-proline rich linker region
joining RZD to NHR2 (amino acids 439-510) did not affect
transcription (FIG. 10). Interestingly, individual NHR1 and NHR2
displayed even stronger repressor activities than the full-length
m-Neu1. These data show that m-Neu1 acts as a transcriptional
repressor when tethered to a promoter via a heterologous DNA
binding domain (DBD). To summarize, m-Neu1 represses the activity
of both TATA and TATA-less promoters in transient expression
assays.
EXAMPLE 5
Nucleocytoplasmic Shuttling of Neu1 Protein
[0154] The function of Neu1 as a transcriptional repressor implies
its nuclear localization. A putative lysine rich nuclear
localization signal (NLS) that is present in d-Neu protein (d-NLS)
is not conserved in mammals. Boulianne et al., EMBO J, 10:2975-2983
(1991) and Price et al., EMBO J, 12:2411-2418 (1993). To study the
subcellular localization of m-Neu1 protein expression constructs
were generated encoding tagged m-Neu-fusion proteins. The
expression of these vectors was analyzed in Neuro2A cells.
[0155] The results from this work is shown in FIG. 11. This figure
shows the subcellular localization of m-Neu1-FLAG and m-Neu1-EGFP
fusion proteins in Neuro2A cells. Neuro2A cells were transfected
expression plasmids encoding m-Neu1-FLAG (A, E, F) and different
m-Neu1-EGFP (B, C, D) fusion proteins or with parental pFLAG (G)
and pEGFP-C3 (H) expression plasmids. Numbers indicate the amino
acids of Neu1 fused to the C-terminal of FLAG and EGFP. Presented
are images of FLAG immunofluorescence using anti-FLAG antibody (A,
E, F, G) and direct fluorescence of EGFP fusion proteins (B, C, D,
H).
[0156] More specifically, A, shows the Neu1-FLAG fusion protein is
localized either in the cytoplasm or in the nucleus; B, shows that
Neu1/120-EGFP fusion protein containing amino acids 1-120 of Neu1
exhibits constitutive nuclear localization in all cells; C, shows
that the Neu1-60/120-EGFP fusion protein is seen exclusively in the
nucleus revealing that the first 60 amino acids of NHR1 are
sufficient for nuclear localization of Neu1; in D, the
Neu1-1/60-EGFP fusion protein containing the N-terminal 60 amino
acids of the brain- specific isoform of m-Neu1 shows localization
that is identical to H, localization of EGFP synthesized from the
parental pEGFP-C3 expression plasmid; E, inhibition of nuclear
export of Neu1-FLAG fusion protein by treatment of Neuro2A cells
with leptomycin B (10 ng/ml) for 12 hours or F, 2 days of RA and
dBcAMP mediated differentiation leads to predominant nuclear
localization of Neu1-FLAG fusion protein; G, FLAG synthesized from
parental plasmid is distributed evenly in the cell.
[0157] The fusion protein m-Neu1-FLAG comprised of the full-length
m-Neu1 with N-terminal FLAG tag showed either predominantly nuclear
(.about.40% of the positive cells) or predominantly cytoplasmic
(.about.60% of the positive cells) distribution. If localized in
the cytoplasm, m-Neu1-FLAG fusion protein was observed in the form
of granular speckles in the perinuclear area, in the vicinity of
plasma membrane and in neurites (FIG. 11A).
[0158] m-Neu1 does not have sequences that are similar to the
lysine-rich d-NLS, however, two smaller clusters of arginine and
lysine rich amino acids (HKAVKAR (SEQ ID NO: 43) at 80-85 and
RLKITKK (SEQ ID NO: 44) at 107-113) are present in the NHR1 of
m-Neu1 (FIG. 1). To investigate whether these putative NLSs are
functional, the subcellular localization of m-Neu1 deletion mutants
fused to the C-terminus of EGFP were analyzed in living cells.
Neu-1/120-EGFP fusion protein containing the first 120 amino acids
of m-Neu1 displayed nuclear localization in virtually all the cells
(100%) (FIG. 11B). Deletion of the first 60 amino acids from the
N-terminal region of m-Neu1 up to the NHR1 domain (Neu-60/120-EGFP)
did not change the predominant nuclear localization of the
Neu-1/120-EGFP fusion protein (FIG. 11C). The N-terminal region of
Neu1 fused to EGFP (Neu1/120-EGFP) displayed overall cellular
distribution of the fusion protein (FIG. 11D) which was identical
to the localization of EGFP (FIG. 11H). These results showed that
the region containing the first 60 amino acids of NHR1 (amino acids
60-120) including two putative NLS domains are sufficient for
nuclear import of m-Neu1.
[0159] Localization of m-Neu1 fusion proteins in the nucleus and
cytoplasm suggest that m-Neu1 transport could be the subject of
regulation. Various proteins have been shown to be exported from
the nucleus by the CRM1/exportin1-related export pathway that is
blocked by the antibiotic leptomycin B (LMB), a specific inhibitor
of nuclear export mediated by leucine-rich nuclear export signals
(NES). Hood and Silver, Curr Opin Cell Biol, 11:241-247 (1999);
Izaurralde and Adam, RNA, 4(:351-364 (1998); Ullman et al., Cell,
90:967-970 (1997); Weiss (1998); Fornerod et al., Cell,
90:1051-1060 (1997); Fukuda et al., Nature, 390:308-311 (1997);
Kudo et al., Exp Cell Res, 242:540-547 (1998); Kudo et al., Proc
Natl Acad Sci USA, 96:9112-9117 (1999); and Nishi et al., J Biol
Chem, 269:6320-6324 (1994). m-Neu1 protein contains two putative
leucine-rich sequences, one in the end of each of the NHR (FIG. 1),
that are similar to the identified NES in different proteins.
Kogerman et al., Nat Cell Bio, 1:312-319 (1999); Taagepera et al.,
Proc Natl Acad Sci USA, 95:7457-7462 (1998); Ullman et al., Cell,
90:967-970 (1997); and Yamaga et al., J Biol Chem, 274:28537-28541
(1999). In view of these results, the effect of LMB on the
localization of m-Neu-FLAG in Neuro2A cells was next studied.
[0160] Treatment of Neuro2A cells transfected with m-Neu1-FLAG
expression construct with LMB (10 ng/ml) resulted in exclusively
nuclear localization of m-Neu1-FLAG at 12 hours post-treatment in
virtually all the cells (FIG. 11E). These results suggest that
m-Neu1 protein shuttles between nucleus and cytoplasm, and that the
CRM1/exportin1-related pathway is involved in nuclear export.
[0161] Interestingly, the number of cells with nuclear localization
of m-Neu1-tagged protein increased substantially (from 45% to 80%)
12 hs after the RA- and cAMP-mediated neuronal differentiation
(FIG. 11F). It suggests that neuronal differentiation changes the
mechanisms that are responsible for the translocation of m-Neu1
protein in Neuro2A cells.
EXAMPLE 6
Characterization of Neu1 Homologs Neu2, Neu3 and Neu4
[0162] Screening of cDNA libraries and RT-PCR amplification has
resulted in isolation of several homologs of mammalian neu1 genes.
Polynucleotide sequences of the following homologs are presented as
SEQ. ID. NOS.: human neu2 cDNA of SEQ. ID. NO.:21; human neu2
alternatively spliced form h-neu2-.DELTA.NHR1 of SEQ. ID. NO.:23;
human neu2 alternatively spliced form h-neu2-.DELTA.NHR2 of SEQ.
ID. NO.:25; rat neu2 cDNA SEQ. ID. NO.: 27; human neu3 cDNA of SEQ.
ID. NO.: 29; mouse neu3 cDNA of SEQ. ID. NO.: 31; and human neu4
cDNA (partial) of SEQ. ID. NO.: 33. Corresponding peptide sequences
are presented as SEQ. ID. NOs: human Neu2 protein of SEQ. ID. NO.:
22; human Neu2 alternatively spliced form h-neu2-.DELTA.NHR1 of
SEQ. ID. NO.:24;; human Neu2 alternatively spliced form
h-neu2-.DELTA.NHR2 of SEQ. ID. NO.:26; rat Neu2 protein SEQ. ID.
NO.: 28; human Neu3 protein of SEQ. ID. NO.: 30; mouse Neu3 protein
of SEQ. ID. NO.: 32; and human Neu4 protein (partial) of SEQ. ID.
NO.: 34. All the mammalian Neu proteins show significant homology
in the NHR and Ring zinc finger domains.
[0163] Alignment of neuralized homology motifs of human Neu1, Neu2,
and Neu3 proteins is shown in FIG. 12. Amino acid sequence
comparison of the neuralized homology regions (NHR) of Drosophila
neu and human neu1, neu2 and neu3 proteins is shown in FIG. 12.
Amino acids that are identical in human and Drosophila neu proteins
are highlighted in white on black background. Amino acids that are
similar in human and Drosophila neu proteins are boxed and
highlighted on grey background. h1I, NHR1 of human neu1; h2I, NHR1
of human neu2; h3, NHR of human neu3; dI, NHR1 of Drosophila neu;
dII, NHR2 of d-neu; h1II, NHR2 of human neu1; h2II, NHR2 of human
neu2.
[0164] Expression of neu1 mRNA is highest in adult, mature neurons
(FIG. 2-7). neu2, in contrast to neu1, is expressed at high levels
already in the embryonic brain and the expression levels decrease
during postnatal development (FIG. 13). Total RNA was isolated from
the indicated mouse and rat brain regions and non-neural tissues
and the levels of neu2 transcripts were analyzed by RNase
protection assays. cRNA probes that were used for detection of neu2
transcripts covered the region encoding the NHR1 of rat neu2.
Specific protected fragments are indicated on the left of the
panel. R-NEU2, rat neu2 transcript; M-NEU2, mouse neu2 transcript.
E, Embryonic day; P, postnatal day; ad, adult; HC, hippocampus;
CTX, cerebral cortex; OLF, olfactory bulb; STR, striatum; THA,
thalamus; HTH, hypothalamus; COL, colliculi; MID, ventral midbrain;
CBL, cerebellum; PONS, pons; MED, medulla;SP.C, spinalcord; PIT,
pituitary; MUS, muscle; SPLCE, spleen tRNA, yeast tRNA as a
negative control.
[0165] neu3 is widely expressed, with highest levels in immune
tissues spleen and thymus, and in the lung (FIG. 14). Total RNA was
isolated from the indicated mouse brain regions and non-neural
tissues and the levels of neu3 transcripts were analyzed by RNase
protection assays. cRNA probes that were used for detection of neu3
transcripts covered the region encoding the NHR of mouse neu3.
Specific protected fragments are indicated on the left of the
panel. E, Embryonic day; P, postnatal day; AD, adult; HC,
hippocampus; CTX, cerebral cortex; OLF, olfactory bulb; STR,
striatum; THA, thalamus; COL, colliculi; MID, midbrain; CBL,
cerebellum; PONS, pons; MED, medulla; LIV, liver; HEA, heart; KID,
kidney; LUN, lung; MUS, muscle; SPL, spleen; TES, testis; THY,
thymus; tRNA, yeast tRNA as a negative control.
[0166] Expression of neu4 was detected only in muscle and
heart.
EXAMPLE 7
Isolation and Characterization of Factors Interacting with Neu1
[0167] An adult rat brain library was screened using a yeast two
hybrid system with a mouse Neu1 protein as a ligand with which to
isolate and identify proteins that interact with Neu1. Fifty-three
(53) clones were isolated. cDNAs that yielded more than 1 clone
were sequenced and identified as four interactors: NeuI-1, NeuI-2,
NeuI-3, and NeuI-4.
[0168] Sequence analyses showed that all interactors were novel
proteins and contain RING finger domain located in the C-terminus
of the protein. NeuI-1 (4 clones) is a novel splice variant (SEQ.
ID. NO.: 29) of zinc finger protein Miz1/PIASX/ARIP3 (GenBank
accession number NM.sub.--008602; AF077953; AF077954; AF044058).
NeuI-2 (3 clones) is a fourth homologue (SEQ. ID. NO.: 30; GenBank
accession number AF277171; AF302084) of zinc finger protein ZNF127
(GenBank accession number U19106; U19107), NeuI-3 (9 clones) has
highest homology to a human hypothetical protein (GenBank accession
number AK001459) and to a Drosophila hypothetical protein
(AAF56052.2) produced from CG4813 gene of a genomic scaffold
(GenBank accession number AE003740) (SEQ. ID. NO.: 31), and NeuI-4
(12 clones) is the homologue of the androgen receptor co-activator
ARA54 (SEQ. ID. NO.: 32; GenBank accession number AF060544).
[0169] Expression of neuI-1-neuI-4 mRNAs was analyzed in developing
and adult rat brain and non-neural tissues (FIG. 15). Total RNA was
isolated from the indicated rat tissues and the levels of
transcripts were analyzed by RNase protection assays with cRNA
probes specific for NeuI-1, NeuI-2, NeuI-3 and NeuI-4 transcripts.
Specific protected fragments are indicated on the left of each
panel. Bottom panel shows the levels of GAPDH mRNA in the RNA
samples. E, Embryonic day; P, postnatal day; ad, adult; cbl,
cerebellum; ctx, cerebral cortex; stem, brainstem; hc, hippocampus;
hc+KA, hippocampus from rats treated for 4h with the glutamate
receptor agonist kainic acid; hea, heart; kid, kidney; mus,
skeletal muscle; spl, spleen; thy, thymus; tes, testis; li, liver;
lu, lung; tRNA, yeast tRNA as a negative control.
[0170] All the identified Neu1 interactors were expressed in the
brain and skeletal muscle, tissues where neu1 mRNA is predominantly
expressed. This suggests that neuI-1-neuI-4 interact with Neu 1
protein and modify its activity in vivo.
EXAMPLE 8
Neu1 Affects the Activation of Immediate Early Genes (IEGs)
[0171] The function of Neu1 as a calcium-dependent transcriptional
repressor was studied by transfecting Neuro2A cells with m-neu1-CMV
expression vector. This system was used to examiner whether or not
Neu1 could suppress transcription of IEGs in response to the
simultaneous stimulation of cells with calcium, ionophore A23187,
and forskolin. As a combination of forskolin with calcium,
ionophores leads to increased c-fos mRNA levels in Swiss 3T3 cells
(Mehmet H, 1990). Because the induction of immediate early genes by
Ca.sup.2+ influx requires cAMP-dependent protein kinase in PC12
cells (Ginty DD, 1991), it was decided to apply the forskolin and
ionophore co-treatment to Neuro-2A cells.
[0172] Upon transient transfection of m-Neu1 into Neuro2A cells,
activation of transcription of several IEGs (c-fos, junB, junD,
c-jun, fra-1, and fra-2) from their endogenous promoters was
significantly reduced in response to the raised intracellular
Ca.sup.2+ (Ca ionophore) and cAMP activity (forskolin) levels. Neu1
elevated levels affected the amplitude of the IEG mRNA induction,
however, had no effect on the time course of induction. This
finding couples Neu1 to stimulus (calcium)-dependent
transcriptional regulation.
EXAMPLE 9
Interaction with TBP
[0173] Because m-Neu1 affects a variety of target gene promoters in
transient transfection assays, it was hypothesized that m-Neu1
functions by interfering directly with the function of Pol II
complex, particularly suppressing the TBP transcriptional activity.
To examine this possibility, m-Neu-CMV expression constructs were
cotransfected together with m-TBP-CMV. The effect of this procedure
on thymidine kinase (tk) promoter was examined. Increasing amounts
of Neu-CMV while mTBP-CMV amounts were kept constant (and vice
versa) resulted in the decreased activity of the reporter gene in
Neuro2A cells. These results suggested that m-Neu1 could repress
transcription by direct interaction with TBP.
EXAMPLE 10
Neoplastic Diagnostic Assay Using Genomic DNA
[0174] A biopsy is obtained from a subject possibly suffering from
a neoplastic disease, such as an astrocytoma. Following excision of
the sample, the tissue is immediately minced and quickly frozen in
liquid nitrogen. A sample 1 gram sample of tissue is then ground
with a prechilled mortar and pestle for suspension. The ground
tissue is then suspended in approximately 1.2 ml of digestions
buffer (100 mM NaCl, 10 mM Tris-Cl, pH 8.0, 25 mM EDTA, pH 8.0,
0.5% (w/v) SDS, and 0.1 mg/ml proteinase K, which is added fresh
for each use). Samples are shaken and incubated for 12 to 18 hours
at 50.degree. C.
[0175] Following the incubation period, the samples are extracted
with an equal volume of phenol/chloroform/isoamyl alcohol, to
remove proteinatious material. Centrifuge the mixture for 10
minutes at 1700.times.g. If phases do not resolve well, add another
volume of digestion buffer, omitting proteinase K, and repeat
centrifugation. Repeat the extraction until no thick white material
appears at the interface. Transfer the top aqueous layer to a new
tube.
[0176] To this tube is added 1/2 volume of 7.5 M ammonium acetate
and 2 volumes of 100% ethanol. This mixture is centrifuged for 2
minutes at 1700.times.g. Following the centrifugation, the
resulting pellet is washed with 70% ethanol, and then air dried and
will be resuspended in Tris-EDTA buffer at approximately 1
mg/ml.
[0177] To prevent shearing of high molecular weight DNA it may be
advisable to remove organic solvents and salt by two dialyses
against 100 volumes of Tris-EDTA buffer for more than 24 hours. If
this step is performed, the pellet is not resuspended in Tris-EDTA
buffer.
[0178] The purified DNA is analyzed for the presence or mutation of
a wild type copy of a neu family gene using Southern Blotting and
sequencing. The absence or mutation of such in the subject
indicates the presence of a malignancy.
EXAMPLE 11
Neoplastic Diagnostic Assay Using RNA Analysis
[0179] A tissue sample is obtained from a subject possibly
suffering from a neoplastic disease. The biopsy is removed and cut
into less than 2 gram pieces. These pieces are quick-frozen in
liquid nitrogen. Twenty (20 ml) of tissue guanidinium solution is
used to process 2 grams of tissue. The tissue guanidinium solution
is prepared by dissolving 590.8 grams of guanidinium isothiocyanate
in approximately 400 ml DEPC-treated H.sub.2O. To this is added 25
ml of 2M Tris-Cl, pH 7.5 (0.05 M final) and 20 ml of 0.5 M
Na.sub.2EDTA, pH 8.0 (0.01 M final). Stir overnight, adjust the
volume to 950 ml, and filter. Finally, add 50 ml 2-ME.
[0180] Once the tissue guanidinium solution is added to the tissue,
the sample is immediately ground in a tissuemizer with two or three
ten second bursts. Following disruption, the solution is subjected
to centrifugation for 10 minutes at 12,000.times.g in a SS-34
rotor, at 12.degree. C. To the supernatant is added 0.1 volumes of
20% Sarkosyl. This mixture is then subjected to heat at 65.degree.
C., for 2 minutes.
[0181] To this heated solution is added 0.1 grams of CsCl/ml of
solution, which is mixed until it dissolves. The sample is next
layered over 9 ml of 5.7M CsCl in silanized and autoclaved SW-28
tubes. These tubes are centrifuged overnight at 113,000.times.g in
a SW-28 rotor at 22.degree. C.
[0182] Following the centrifugation step, the supernatant is
removed and the tubes are inverted to drain. The bottom of each
tube is removed and the RNA pelleted contained therein is placed in
a 50-ml plastic tube. Three (3) ml of tissue resuspension buffer
and allow pellet to resuspend overnight or longer at 4.degree. C.
Extract solution sequestially with 25:24:1
phenol/chloroform/isoamyl alcohol, then with 24:1
chloroform/isoamyl alcohol. Add 0.1 volume of 3 M sodium acetate,
pH 5.2, and 2.5 volume of 100% ethanol, precipitate, and resuspend
RNA in water. The sample is quantitated and analyzed for the
expression of the neu family of genes. Samples with aberent
expression levels indicate the presence of neoplastic cells.
EXAMPLE 12
Ex Vivo Exogenous Gene Expression
[0183] Cells are isolated from a subject to be transfected with a
construct encoding Neu protein (Neu construct). The Neu construct
is transfected using DEAE-dextran. The cells are seeded in 6-well
tissue culture plates and are transfected with a total of 1-2 .mu.g
of total DNA containing the Neu contruct. After 5 hours, 1 ml of
DMEM containing 20% fetal bovine serum is added and the cells are
allowed to incubate overnight.
[0184] Expression of Neu is then determined using
immunoprecipitation with an antibody-agarose conjugate (8 .mu.g)
plus 25 .mu.l of a 50% slurry of Protein A-agarose. Immune
complexes are washed in a wash buffer (20 mM Hepes, pH 7.4, 10 mM
MgCl.sub.2, and 1 mM DTT). The immune complexes are placed in a
denaturing protein sample buffer that separated the antibody from
any bound antigen. The protein samples are then run on
SDS-polyacrylamide gel electrophoresis to detect the expression of
a target Neu protein.
[0185] The foregoing description details certain embodiments of the
invention. It will be appreciated, however, that no matter how
detailed the foregoing appears in the text, the invention can be
practised in many ways. Although this invention has been described
in terms of certain preferred embodiments, other embodiments which
will be apparent to those of ordinary skill in the art in view of
the disclosure herein are also within the scope of this invention.
As is also stated above, it should be noted that the use of
particular terminology when describing certain features or aspects
of the invention should not be taken to imply that the terminology
is being re-defined herein to be restricted to include any specific
characteristics of the features or aspects of the invention with
which that terminology is associated. Accordingly, the scope of the
invention should therefore be construed in accordance with the
appended claims and any equivalents thereof.
Sequence CWU 1
1
48 1 1725 DNA Homo sapien 1 atgggtaaca acttctccag tatcccctcg
ctgccccgag gaaacccgag ccgcgcgccg 60 cggggccacc cccagaacct
caaagactct atcgggggcc ccttccccgt cacttctcac 120 cgatgccacc
acaagcagaa gcactgtccg gcagtgctgc ccagcggggg gctcccagcc 180
acgccgctgc tcttccaccc gcacaccaag ggctcccaga tcctcatgga cctcagccac
240 aaggctgtca agaggcaggc cagcttctgc aacgccatca ccttcagcaa
ccgcccggtc 300 ctcatctacg agcaagtcag gctgaagatc accaagaagc
agtgctgctg gagcggggcc 360 ctgcggctgg gcttcaccag caaggacccg
tcccgcatcc accctgactc gctgcccaag 420 tacgcctgcc ccgacctggt
gtcccagagt ggcttctggg ccaaggcgct gcctgaggag 480 tttgccaatg
agggcaacat catcgcattc tgggtggaca agaagggccg tgtcttccac 540
cgcatcaacg actcggctgt tatgctgttc ttcagcgggg tccgcacggc cgacccgctc
600 tgggccctgg tggacgtcta cggcctcacg cggggcgtcc agctgcttga
tagcgagctg 660 gtgctcccgg actgtctgcg gccgcgctcc ttcaccgccc
tgcggcggcc gtcgctgcgg 720 cgcgaggcgg acgacgcgcg cctctcggtg
agcctatgcg acctcaacgt gccgggcgcg 780 gacggcgacg aggccgcgcc
ggccgccggc tgccccatcc cgcagaactc actcaactcg 840 cagcacagcc
gcgcgctgcc ggcgcagctc gacggcgacc tgcgtttcca cgccctgcgc 900
gccggcgcgc acgtccgcat cctcgacgag cagacggtgg cgcgcgtgga gcacgggcgc
960 gacgagcgcg cgctcgtctt caccagccgg cccgtgcgcg tggccgagac
catcttcgtc 1020 aaggtcacgc gctcgggtgg cgcgcggccc ggcgcgctgt
cgttcggcgt caccacgtgc 1080 gaccccggca cgctgcggcc ggccgacctg
cctttcagcc ctgaggccct ggtggaccgc 1140 aaggaattct gggccgtgtg
ccgcgtgccc gggcccctgc acagcggcga catcctgggc 1200 ctggtggtca
acgccgacgg cgagctgcac ctcagccaca atggcgcggc cgccggcatg 1260
cagctgtgcg tggacgcctc gcagccgctt tggatgctct tcggcctgca cgggaccatc
1320 acgcagatcc gcatcctcgg ctccactatc ctggccgagc ggggtatccc
gtcactcccc 1380 tgctcccctg cctccacgcc aacctcgccc agtgccctgg
gcagccgcct gtctgacccc 1440 ttgctcagca cgtgcagctc tggccctctg
ggtagctctg ctggtgggac agcccccaat 1500 tcgccagtga gcctgcccga
gtcgccagtg accccaggtc tgggccagtg gagcgatgag 1560 tgcaccattt
gctatgaaca cgcggtggac acggtcatct acacatgtgg ccacatgtgc 1620
ctctgctacg cctgtggcct gcgcctcaag aaggctctgc acgcctgctg ccccatctgc
1680 cgccgcccca tcaaggacat catcaagacc taccgcagct cctag 1725 2 574
PRT Homo sapien 2 Met Gly Asn Asn Phe Ser Ser Ile Pro Ser Leu Pro
Arg Gly Asn Pro 1 5 10 15 Ser Arg Ala Pro Arg Gly His Pro Gln Asn
Leu Lys Asp Ser Ile Gly 20 25 30 Gly Pro Phe Pro Val Thr Ser His
Arg Cys His His Lys Gln Lys His 35 40 45 Cys Pro Ala Val Leu Pro
Ser Gly Gly Leu Pro Ala Thr Pro Leu Leu 50 55 60 Phe His Pro His
Thr Lys Gly Ser Gln Ile Leu Met Asp Leu Ser His 65 70 75 80 Lys Ala
Val Lys Arg Gln Ala Ser Phe Cys Asn Ala Ile Thr Phe Ser 85 90 95
Asn Arg Pro Val Leu Ile Tyr Glu Gln Val Arg Leu Lys Ile Thr Lys 100
105 110 Lys Gln Cys Cys Trp Ser Gly Ala Leu Arg Leu Gly Phe Thr Ser
Lys 115 120 125 Asp Pro Ser Arg Ile His Pro Asp Ser Leu Pro Lys Tyr
Ala Cys Pro 130 135 140 Asp Leu Val Ser Gln Ser Gly Phe Trp Ala Lys
Ala Leu Pro Glu Glu 145 150 155 160 Phe Ala Asn Glu Gly Asn Ile Ile
Ala Phe Trp Val Asp Lys Lys Gly 165 170 175 Arg Val Phe His Arg Ile
Asn Asp Ser Ala Val Met Leu Phe Phe Ser 180 185 190 Gly Val Arg Thr
Ala Asp Pro Leu Trp Ala Leu Val Asp Val Tyr Gly 195 200 205 Leu Thr
Arg Gly Val Gln Leu Leu Asp Ser Glu Leu Val Leu Pro Asp 210 215 220
Cys Leu Arg Pro Arg Ser Phe Thr Ala Leu Arg Arg Pro Ser Leu Arg 225
230 235 240 Arg Glu Ala Asp Asp Ala Arg Leu Ser Val Ser Leu Cys Asp
Leu Asn 245 250 255 Val Pro Gly Ala Asp Gly Asp Glu Ala Ala Pro Ala
Ala Gly Cys Pro 260 265 270 Ile Pro Gln Asn Ser Leu Asn Ser Gln His
Ser Arg Ala Leu Pro Ala 275 280 285 Gln Leu Asp Gly Asp Leu Arg Phe
His Ala Leu Arg Ala Gly Ala His 290 295 300 Val Arg Ile Leu Asp Glu
Gln Thr Val Ala Arg Val Glu His Gly Arg 305 310 315 320 Asp Glu Arg
Ala Leu Val Phe Thr Ser Arg Pro Val Arg Val Ala Glu 325 330 335 Thr
Ile Phe Val Lys Val Thr Arg Ser Gly Gly Ala Arg Pro Gly Ala 340 345
350 Leu Ser Phe Gly Val Thr Thr Cys Asp Pro Gly Thr Leu Arg Pro Ala
355 360 365 Asp Leu Pro Phe Ser Pro Glu Ala Leu Val Asp Arg Lys Glu
Phe Trp 370 375 380 Ala Val Cys Arg Val Pro Gly Pro Leu His Ser Gly
Asp Ile Leu Gly 385 390 395 400 Leu Val Val Asn Ala Asp Gly Glu Leu
His Leu Ser His Asn Gly Ala 405 410 415 Ala Ala Gly Met Gln Leu Cys
Val Asp Ala Ser Gln Pro Leu Trp Met 420 425 430 Leu Phe Gly Leu His
Gly Thr Ile Thr Gln Ile Arg Ile Leu Gly Ser 435 440 445 Thr Ile Leu
Ala Glu Arg Gly Ile Pro Ser Leu Pro Cys Ser Pro Ala 450 455 460 Ser
Thr Pro Thr Ser Pro Ser Ala Leu Gly Ser Arg Leu Ser Asp Pro 465 470
475 480 Leu Leu Ser Thr Cys Ser Ser Gly Pro Leu Gly Ser Ser Ala Gly
Gly 485 490 495 Thr Ala Pro Asn Ser Pro Val Ser Leu Pro Glu Ser Pro
Val Thr Pro 500 505 510 Gly Leu Gly Gln Trp Ser Asp Glu Cys Thr Ile
Cys Tyr Glu His Ala 515 520 525 Val Asp Thr Val Ile Tyr Thr Cys Gly
His Met Cys Leu Cys Tyr Ala 530 535 540 Cys Gly Leu Arg Leu Lys Lys
Ala Leu His Ala Cys Cys Pro Ile Cys 545 550 555 560 Arg Arg Pro Ile
Lys Asp Ile Ile Lys Thr Tyr Arg Ser Ser 565 570 3 1674 DNA Homo
sapien 3 atggggggac agatcacccg gagcactctc cacgactcta tcgggggccc
cttccccgtc 60 acttctcacc gatgccacca caagcagaag cactgtccgg
cagtgctgcc cagcgggggg 120 ctcccagcca cgccgctgct cttccacccg
cacaccaagg gctcccagat cctcatggac 180 ctcagccaca aggctgtcaa
gaggcaggcc agcttctgca acgccatcac cttcagcaac 240 cgcccggtcc
tcatctacga gcaagtcagg ctgaagatca ccaagaagca gtgctgctgg 300
agcggggccc tgcggctggg cttcaccagc aaggacccgt cccgcatcca ccctgactcg
360 ctgcccaagt acgcctgccc cgacctggtg tcccagagtg gcttctgggc
caaggcgctg 420 cctgaggagt ttgccaatga gggcaacatc atcgcattct
gggtggacaa gaagggccgt 480 gtcttccacc gcatcaacga ctcggctgtt
atgctgttct tcagcggggt ccgcacggcc 540 gacccgctct gggccctggt
ggacgtctac ggcctcacgc ggggcgtcca gctgcttgat 600 agcgagctgg
tgctcccgga ctgtctgcgg ccgcgctcct tcaccgccct gcggcggccg 660
tcgctgcggc gcgaggcgga cgacgcgcgc ctctcggtga gcctatgcga cctcaacgtg
720 ccgggcgcgg acggcgacga ggccgcgccg gccgccggct gccccatccc
gcagaactca 780 ctcaactcgc agcacagccg cgcgctgccg gcgcagctcg
acggcgacct gcgtttccac 840 gccctgcgcg ccggcgcgca cgtccgcatc
ctcgacgagc agacggtggc gcgcgtggag 900 cacgggcgcg acgagcgcgc
gctcgtcttc accagccggc ccgtgcgcgt ggccgagacc 960 atcttcgtca
aggtcacgcg ctcgggtggc gcgcggcccg gcgcgctgtc gttcggcgtc 1020
accacgtgcg accccggcac gctgcggccg gccgacctgc ctttcagccc tgaggccctg
1080 gtggaccgca aggaattctg ggccgtgtgc cgcgtgcccg ggcccctgca
cagcggcgac 1140 atcctgggcc tggtggtcaa cgccgacggc gagctgcacc
tcagccacaa tggcgcggcc 1200 gccggcatgc agctgtgcgt ggacgcctcg
cagccgcttt ggatgctctt cggcctgcac 1260 gggaccatca cgcagatccg
catcctcggc tccactatcc tggccgagcg gggtatcccg 1320 tcactcccct
gctcccctgc ctccacgcca acctcgccca gtgccctggg cagccgcctg 1380
tctgacccct tgctcagcac gtgcagctct ggccctctgg gtagctctgc tggtgggaca
1440 gcccccaatt cgccagtgag cctgcccgag tcgccagtga ccccaggtct
gggccagtgg 1500 agcgatgagt gcaccatttg ctatgaacac gcggtggaca
cggtcatcta cacatgtggc 1560 cacatgtgcc tctgctacgc ctgtggcctg
cgcctcaaga aggctctgca cgcctgctgc 1620 cccatctgcc gccgccccat
caaggacatc atcaagacct accgcagctc ctag 1674 4 557 PRT Homo sapien 4
Met Gly Gly Gln Ile Thr Arg Ser Thr Leu His Asp Ser Ile Gly Gly 1 5
10 15 Pro Phe Pro Val Thr Ser His Arg Cys His His Lys Gln Lys His
Cys 20 25 30 Pro Ala Val Leu Pro Ser Gly Gly Leu Pro Ala Thr Pro
Leu Leu Phe 35 40 45 His Pro His Thr Lys Gly Ser Gln Ile Leu Met
Asp Leu Ser His Lys 50 55 60 Ala Val Lys Arg Gln Ala Ser Phe Cys
Asn Ala Ile Thr Phe Ser Asn 65 70 75 80 Arg Pro Val Leu Ile Tyr Glu
Gln Val Arg Leu Lys Ile Thr Lys Lys 85 90 95 Gln Cys Cys Trp Ser
Gly Ala Leu Arg Leu Gly Phe Thr Ser Lys Asp 100 105 110 Pro Ser Arg
Ile His Pro Asp Ser Leu Pro Lys Tyr Ala Cys Pro Asp 115 120 125 Leu
Val Ser Gln Ser Gly Phe Trp Ala Lys Ala Leu Pro Glu Glu Phe 130 135
140 Ala Asn Glu Gly Asn Ile Ile Ala Phe Trp Val Asp Lys Lys Gly Arg
145 150 155 160 Val Phe His Arg Ile Asn Asp Ser Ala Val Met Leu Phe
Phe Ser Gly 165 170 175 Val Arg Thr Ala Asp Pro Leu Trp Ala Leu Val
Asp Val Tyr Gly Leu 180 185 190 Thr Arg Gly Val Gln Leu Leu Asp Ser
Glu Leu Val Leu Pro Asp Cys 195 200 205 Leu Arg Pro Arg Ser Phe Thr
Ala Leu Arg Arg Pro Ser Leu Arg Arg 210 215 220 Glu Ala Asp Asp Ala
Arg Leu Ser Val Ser Leu Cys Asp Leu Asn Val 225 230 235 240 Pro Gly
Ala Asp Gly Asp Glu Ala Ala Pro Ala Ala Gly Cys Pro Ile 245 250 255
Pro Gln Asn Ser Leu Asn Ser Gln His Ser Arg Ala Leu Pro Ala Gln 260
265 270 Leu Asp Gly Asp Leu Arg Phe His Ala Leu Arg Ala Gly Ala His
Val 275 280 285 Arg Ile Leu Asp Glu Gln Thr Val Ala Arg Val Glu His
Gly Arg Asp 290 295 300 Glu Arg Ala Leu Val Phe Thr Ser Arg Pro Val
Arg Val Ala Glu Thr 305 310 315 320 Ile Phe Val Lys Val Thr Arg Ser
Gly Gly Ala Arg Pro Gly Ala Leu 325 330 335 Ser Phe Gly Val Thr Thr
Cys Asp Pro Gly Thr Leu Arg Pro Ala Asp 340 345 350 Leu Pro Phe Ser
Pro Glu Ala Leu Val Asp Arg Lys Glu Phe Trp Ala 355 360 365 Val Cys
Arg Val Pro Gly Pro Leu His Ser Gly Asp Ile Leu Gly Leu 370 375 380
Val Val Asn Ala Asp Gly Glu Leu His Leu Ser His Asn Gly Ala Ala 385
390 395 400 Ala Gly Met Gln Leu Cys Val Asp Ala Ser Gln Pro Leu Trp
Met Leu 405 410 415 Phe Gly Leu His Gly Thr Ile Thr Gln Ile Arg Ile
Leu Gly Ser Thr 420 425 430 Ile Leu Ala Glu Arg Gly Ile Pro Ser Leu
Pro Cys Ser Pro Ala Ser 435 440 445 Thr Pro Thr Ser Pro Ser Ala Leu
Gly Ser Arg Leu Ser Asp Pro Leu 450 455 460 Leu Ser Thr Cys Ser Ser
Gly Pro Leu Gly Ser Ser Ala Gly Gly Thr 465 470 475 480 Ala Pro Asn
Ser Pro Val Ser Leu Pro Glu Ser Pro Val Thr Pro Gly 485 490 495 Leu
Gly Gln Trp Ser Asp Glu Cys Thr Ile Cys Tyr Glu His Ala Val 500 505
510 Asp Thr Val Ile Tyr Thr Cys Gly His Met Cys Leu Cys Tyr Ala Cys
515 520 525 Gly Leu Arg Leu Lys Lys Ala Leu His Ala Cys Cys Pro Ile
Cys Arg 530 535 540 Arg Pro Ile Lys Asp Ile Ile Lys Thr Tyr Arg Ser
Ser 545 550 555 5 1161 DNA Homo sapien 5 atgggtaaca acttctccag
tatcccctcg ctgccccgag gaaacccgag ccgcgcgccg 60 cggggccacc
cccagaacct caaagatagc gagctggtgc tcccggactg tctgcggccg 120
cgctccttca ccgccctgcg gcggccgtcg ctgcggcgcg aggcggacga cgcgcgcctc
180 tcggtgagcc tatgcgacct caacgtgccg ggcgcggacg gcgacgaggc
cgcgccggcc 240 gccggctgcc ccatcccgca gaactcactc aactcgcagc
acagccgcgc gctgccggcg 300 cagctcgacg gcgacctgcg tttccacgcc
ctgcgcgccg gcgcgcacgt ccgcatcctc 360 gacgagcaga cggtggcgcg
cgtggagcac gggcgcgacg agcgcgcgct cgtcttcacc 420 agccggcccg
tgcgcgtggc cgagaccatc ttcgtcaagg tcacgcgctc gggtggcgcg 480
cggcccggcg cgctgtcgtt cggcgtcacc acgtgcgacc ccggcacgct gcggccggcc
540 gacctgcctt tcagccctga ggccctggtg gaccgcaagg aattctgggc
cgtgtgccgc 600 gtgcccgggc ccctgcacag cggcgacatc ctgggcctgg
tggtcaacgc cgacggcgag 660 ctgcacctca gccacaatgg cgcggccgcc
ggcatgcagc tgtgcgtgga cgcctcgcag 720 ccgctttgga tgctcttcgg
cctgcacggg accatcacgc agatccgcat cctcggctcc 780 actatcctgg
ccgagcgggg tatcccgtca ctcccctgct cccctgcctc cacgccaacc 840
tcgcccagtg ccctgggcag ccgcctgtct gaccccttgc tcagcacgtg cagctctggc
900 cctctgggta gctctgctgg tgggacagcc cccaattcgc cagtgagcct
gcccgagtcg 960 ccagtgaccc caggtctggg ccagtggagc gatgagtgca
ccatttgcta tgaacacgcg 1020 gtggacacgg tcatctacac atgtggccac
atgtgcctct gctacgcctg tggcctgcgc 1080 ctcaagaagg ctctgcacgc
ctgctgcccc atctgccgcc gccccatcaa ggacatcatc 1140 aagacctacc
gcagctccta g 1161 6 386 PRT Homo sapien 6 Met Gly Asn Asn Phe Ser
Ser Ile Pro Ser Leu Pro Arg Gly Asn Pro 1 5 10 15 Ser Arg Ala Pro
Arg Gly His Pro Gln Asn Leu Lys Asp Ser Glu Leu 20 25 30 Val Leu
Pro Asp Cys Leu Arg Pro Arg Ser Phe Thr Ala Leu Arg Arg 35 40 45
Pro Ser Leu Arg Arg Glu Ala Asp Asp Ala Arg Leu Ser Val Ser Leu 50
55 60 Cys Asp Leu Asn Val Pro Gly Ala Asp Gly Asp Glu Ala Ala Pro
Ala 65 70 75 80 Ala Gly Cys Pro Ile Pro Gln Asn Ser Leu Asn Ser Gln
His Ser Arg 85 90 95 Ala Leu Pro Ala Gln Leu Asp Gly Asp Leu Arg
Phe His Ala Leu Arg 100 105 110 Ala Gly Ala His Val Arg Ile Leu Asp
Glu Gln Thr Val Ala Arg Val 115 120 125 Glu His Gly Arg Asp Glu Arg
Ala Leu Val Phe Thr Ser Arg Pro Val 130 135 140 Arg Val Ala Glu Thr
Ile Phe Val Lys Val Thr Arg Ser Gly Gly Ala 145 150 155 160 Arg Pro
Gly Ala Leu Ser Phe Gly Val Thr Thr Cys Asp Pro Gly Thr 165 170 175
Leu Arg Pro Ala Asp Leu Pro Phe Ser Pro Glu Ala Leu Val Asp Arg 180
185 190 Lys Glu Phe Trp Ala Val Cys Arg Val Pro Gly Pro Leu His Ser
Gly 195 200 205 Asp Ile Leu Gly Leu Val Val Asn Ala Asp Gly Glu Leu
His Leu Ser 210 215 220 His Asn Gly Ala Ala Ala Gly Met Gln Leu Cys
Val Asp Ala Ser Gln 225 230 235 240 Pro Leu Trp Met Leu Phe Gly Leu
His Gly Thr Ile Thr Gln Ile Arg 245 250 255 Ile Leu Gly Ser Thr Ile
Leu Ala Glu Arg Gly Ile Pro Ser Leu Pro 260 265 270 Cys Ser Pro Ala
Ser Thr Pro Thr Ser Pro Ser Ala Leu Gly Ser Arg 275 280 285 Leu Ser
Asp Pro Leu Leu Ser Thr Cys Ser Ser Gly Pro Leu Gly Ser 290 295 300
Ser Ala Gly Gly Thr Ala Pro Asn Ser Pro Val Ser Leu Pro Glu Ser 305
310 315 320 Pro Val Thr Pro Gly Leu Gly Gln Trp Ser Asp Glu Cys Thr
Ile Cys 325 330 335 Tyr Glu His Ala Val Asp Thr Val Ile Tyr Thr Cys
Gly His Met Cys 340 345 350 Leu Cys Tyr Ala Cys Gly Leu Arg Leu Lys
Lys Ala Leu His Ala Cys 355 360 365 Cys Pro Ile Cys Arg Arg Pro Ile
Lys Asp Ile Ile Lys Thr Tyr Arg 370 375 380 Ser Ser 385 7 1749 DNA
mouse 7 atgggtaaca acttctccag tgtctcctct ctgcagcgag gaaacccgag
ccgcgcgtcg 60 cggggccacc cccagaacct caaagaatcc atcgggggct
ccttcccggt gccctctcac 120 cgatgccatc acaagcagaa gcattgcccg
cctacgctgt caggtggggg gctcccggcc 180 acgccgctgc tcttccaccc
ccacactaag ggctcccaga tcctcatgga cctcagccac 240 aaggccgtca
agaggcaggc cagcttctgc aatgccatca ccttcagtaa ccgcccggtg 300
ctcatctacg agcaagtcag gctgaagatc accaagaagc aatgctgctg gagcggggcc
360 ctgcgacttg gcttcaccag caaggaccct tcccgcatcc accccgactc
gctgcccaag 420 tacgcctgcc ctgacctggt gtctcagagt ggcttctggg
ccaaagcatt gcctgaggag 480 tttgccaacg agggcaacat cattgccttc
tgggtggaca agaagggccg cgtcttctac 540 cggatcaatg agtcagctgc
tatgcttttc ttcagtgggg tccggacggt ggacccgctc 600 tgggccctgg
tggacgtcta cggcctcacg cggggtgtcc agctgctaga cagcgagctg 660
gtgctgcccg actgcctgcg gccgcgctcc ttcaccgcgc tgcggcggcc gtcgctgcgg
720 tgcgaggcgg atgaggcgcg cctgtcggtg agcctgtgcg acctcaacgt
gccgggagcc 780 gacggcgacg acggcgcacc gcctgccggc tgcccgatcc
cgcagaactc gctcaattct 840 cagcacagcc gcgcgctgcc ggcgcagctc
gacggcgacc tgcgcttcca cgcgcttcgc 900 gccggcgcgc acgtccgcat
cctggacgag cagacggtgg cgcgcctgga gcacgggcgc 960 gacgagcgcg
cgctcgtctt caccagccgg cctgtgagcg
tggccgagac catcttcatc 1020 aaggtcacgc gctcgggcgg ggggcgagcg
ggcgcgctgt ccttcggggt caccacgtgt 1080 gaccctggca cgctgcggcc
cgcggacctg cccttcagcc ccgaggccct ggtggaccgc 1140 aaggagttct
gggcggtgtg tcgcgtgccc gggcctctgc acagcggcga catcctgggc 1200
ctggtggtca acgcggacgg agagctgcac ctgagtcaca acggcgcggc ggccggcatg
1260 cagctgtgcg tggatgcctc gcagcccctc tggatgctct tcagcctgca
tggcgccatc 1320 acgcaggtcc gcatcctcgg ctccaccatc atgactgaac
ggggtggccc atctctcccc 1380 tgctcacctg cttccactcc aacctcaccc
agtgccctgg gcatccgctt gtctgacccc 1440 ttgctcagca cctgcggttc
tgggccccta ggtggctctg ctggagggac agcccccaat 1500 tcacctgtga
gcctgcccga gtcaccggtg accccaggtc tgggccagtg gagtgatgaa 1560
tgcaccattt gctatgaaca cgcagtggat acagtcatct acacgtgtgg ccacatgtgc
1620 ctgtgctact cctgtggcct gcgcctcaag aaggccctgc acgcctgctg
ccccatctgc 1680 cgtcgcccca tcaaggacat catcaagacc taccgcagct
cgtagcccac tgcagagccc 1740 cacctgcac 1749 8 574 PRT mouse 8 Met Gly
Asn Asn Phe Ser Ser Val Ser Ser Leu Gln Arg Gly Asn Pro 1 5 10 15
Ser Arg Ala Ser Arg Gly His Pro Gln Asn Leu Lys Glu Ser Ile Gly 20
25 30 Gly Ser Phe Pro Val Pro Ser His Arg Cys His His Lys Gln Lys
His 35 40 45 Cys Pro Pro Thr Leu Ser Gly Gly Gly Leu Pro Ala Thr
Pro Leu Leu 50 55 60 Phe His Pro His Thr Lys Gly Ser Gln Ile Leu
Met Asp Leu Ser His 65 70 75 80 Lys Ala Val Lys Arg Gln Ala Ser Phe
Cys Asn Ala Ile Thr Phe Ser 85 90 95 Asn Arg Pro Val Leu Ile Tyr
Glu Gln Val Arg Leu Lys Ile Thr Lys 100 105 110 Lys Gln Cys Cys Trp
Ser Gly Ala Leu Arg Leu Gly Phe Thr Ser Lys 115 120 125 Asp Pro Ser
Arg Ile His Pro Asp Ser Leu Pro Lys Tyr Ala Cys Pro 130 135 140 Asp
Leu Val Ser Gln Ser Gly Phe Trp Ala Lys Ala Leu Pro Glu Glu 145 150
155 160 Phe Ala Asn Glu Gly Asn Ile Ile Ala Phe Trp Val Asp Lys Lys
Gly 165 170 175 Arg Val Phe Tyr Arg Ile Asn Glu Ser Ala Ala Met Leu
Phe Phe Ser 180 185 190 Gly Val Arg Thr Val Asp Pro Leu Trp Ala Leu
Val Asp Val Tyr Gly 195 200 205 Leu Thr Arg Gly Val Gln Leu Leu Asp
Ser Glu Leu Val Leu Pro Asp 210 215 220 Cys Leu Arg Pro Arg Ser Phe
Thr Ala Leu Arg Arg Pro Ser Leu Arg 225 230 235 240 Cys Glu Ala Asp
Glu Ala Arg Leu Ser Val Ser Leu Cys Asp Leu Asn 245 250 255 Val Pro
Gly Ala Asp Gly Asp Asp Gly Ala Pro Pro Ala Gly Cys Pro 260 265 270
Ile Pro Gln Asn Ser Leu Asn Ser Gln His Ser Arg Ala Leu Pro Ala 275
280 285 Gln Leu Asp Gly Asp Leu Arg Phe His Ala Leu Arg Ala Gly Ala
His 290 295 300 Val Arg Ile Leu Asp Glu Gln Thr Val Ala Arg Leu Glu
His Gly Arg 305 310 315 320 Asp Glu Arg Ala Leu Val Phe Thr Ser Arg
Pro Val Ser Val Ala Glu 325 330 335 Thr Ile Phe Ile Lys Val Thr Arg
Ser Gly Gly Gly Arg Ala Gly Ala 340 345 350 Leu Ser Phe Gly Val Thr
Thr Cys Asp Pro Gly Thr Leu Arg Pro Ala 355 360 365 Asp Leu Pro Phe
Ser Pro Glu Ala Leu Val Asp Arg Lys Glu Phe Trp 370 375 380 Ala Val
Cys Arg Val Pro Gly Pro Leu His Ser Gly Asp Ile Leu Gly 385 390 395
400 Leu Val Val Asn Ala Asp Gly Glu Leu His Leu Ser His Asn Gly Ala
405 410 415 Ala Ala Gly Met Gln Leu Cys Val Asp Ala Ser Gln Pro Leu
Trp Met 420 425 430 Leu Phe Ser Leu His Gly Ala Ile Thr Gln Val Arg
Ile Leu Gly Ser 435 440 445 Thr Ile Met Thr Glu Arg Gly Gly Pro Ser
Leu Pro Cys Ser Pro Ala 450 455 460 Ser Thr Pro Thr Ser Pro Ser Ala
Leu Gly Ile Arg Leu Ser Asp Pro 465 470 475 480 Leu Leu Ser Thr Cys
Gly Ser Gly Pro Leu Gly Gly Ser Ala Gly Gly 485 490 495 Thr Ala Pro
Asn Ser Pro Val Ser Leu Pro Glu Ser Pro Val Thr Pro 500 505 510 Gly
Leu Gly Gln Trp Ser Asp Glu Cys Thr Ile Cys Tyr Glu His Ala 515 520
525 Val Asp Thr Val Ile Tyr Thr Cys Gly His Met Cys Leu Cys Tyr Ser
530 535 540 Cys Gly Leu Arg Leu Lys Lys Ala Leu His Ala Cys Cys Pro
Ile Cys 545 550 555 560 Arg Arg Pro Ile Lys Asp Ile Ile Lys Thr Tyr
Arg Ser Ser 565 570 9 1698 DNA mouse 9 atggggggac agatcacaag
gaacactatc cacgactcca tcgggggctc cttcccggtg 60 ccctctcacc
gatgccatca caagcagaag cattgcccgc ctacgctgtc aggtgggggg 120
ctcccggcca cgccgctgct cttccacccc cacactaagg gctcccagat cctcatggac
180 ctcagccaca aggccgtcaa gaggcaggcc agcttctgca atgccatcac
cttcagtaac 240 cgcccggtgc tcatctacga gcaagtcagg ctgaagatca
ccaagaagca atgctgctgg 300 agcggggccc tgcgacttgg cttcaccagc
aaggaccctt cccgcatcca ccccgactcg 360 ctgcccaagt acgcctgccc
tgacctggtg tctcagagtg gcttctgggc caaagcattg 420 cctgaggagt
ttgccaacga gggcaacatc attgccttct gggtggacaa gaagggccgc 480
gtcttctacc ggatcaatga gtcagctgct atgcttttct tcagtggggt ccggacggtg
540 gacccgctct gggccctggt ggacgtctac ggcctcacgc ggggtgtcca
gctgctagac 600 agcgagctgg tgctgcccga ctgcctgcgg ccgcgctcct
tcaccgcgct gcggcggccg 660 tcgctgcggt gcgaggcgga tgaggcgcgc
ctgtcggtga gcctgtgcga cctcaacgtg 720 ccgggagccg acggcgacga
cggcgcaccg cctgccggct gcccgatccc gcagaactcg 780 ctcaattctc
agcacagccg cgcgctgccg gcgcagctcg acggcgacct gcgcttccac 840
gcgcttcgcg ccggcgcgca cgtccgcatc ctggacgagc agacggtggc gcgcctggag
900 cacgggcgcg acgagcgcgc gctcgtcttc accagccggc ctgtgagcgt
ggccgagacc 960 atcttcatca aggtcacgcg ctcgggcggg gggcgagcgg
gcgcgctgtc cttcggggtc 1020 accacgtgtg accctggcac gctgcggccc
gcggacctgc ccttcagccc cgaggccctg 1080 gtggaccgca aggagttctg
ggcggtgtgt cgcgtgcccg ggcctctgca cagcggcgac 1140 atcctgggcc
tggtggtcaa cgcggacgga gagctgcacc tgagtcacaa cggcgcggcg 1200
gccggcatgc agctgtgcgt ggatgcctcg cagcccctct ggatgctctt cagcctgcat
1260 ggcgccatca cgcaggtccg catcctcggc tccaccatca tgactgaacg
gggtggccca 1320 tctctcccct gctcacctgc ttccactcca acctcaccca
gtgccctggg catccgcttg 1380 tctgacccct tgctcagcac ctgcggttct
gggcccctag gtggctctgc tggagggaca 1440 gcccccaatt cacctgtgag
cctgcccgag tcaccggtga ccccaggtct gggccagtgg 1500 agtgatgaat
gcaccatttg ctatgaacac gcagtggata cagtcatcta cacgtgtggc 1560
cacatgtgcc tgtgctactc ctgtggcctg cgcctcaaga aggccctgca cgcctgctgc
1620 cccatctgcc gtcgccccat caaggacatc atcaagacct accgcagctc
gtagcccact 1680 gcagagcccc acctgcac 1698 10 557 PRT mouse 10 Met
Gly Gly Gln Ile Thr Arg Asn Thr Ile His Asp Ser Ile Gly Gly 1 5 10
15 Ser Phe Pro Val Pro Ser His Arg Cys His His Lys Gln Lys His Cys
20 25 30 Pro Pro Thr Leu Ser Gly Gly Gly Leu Pro Ala Thr Pro Leu
Leu Phe 35 40 45 His Pro His Thr Lys Gly Ser Gln Ile Leu Met Asp
Leu Ser His Lys 50 55 60 Ala Val Lys Arg Gln Ala Ser Phe Cys Asn
Ala Ile Thr Phe Ser Asn 65 70 75 80 Arg Pro Val Leu Ile Tyr Glu Gln
Val Arg Leu Lys Ile Thr Lys Lys 85 90 95 Gln Cys Cys Trp Ser Gly
Ala Leu Arg Leu Gly Phe Thr Ser Lys Asp 100 105 110 Pro Ser Arg Ile
His Pro Asp Ser Leu Pro Lys Tyr Ala Cys Pro Asp 115 120 125 Leu Val
Ser Gln Ser Gly Phe Trp Ala Lys Ala Leu Pro Glu Glu Phe 130 135 140
Ala Asn Glu Gly Asn Ile Ile Ala Phe Trp Val Asp Lys Lys Gly Arg 145
150 155 160 Val Phe Tyr Arg Ile Asn Glu Ser Ala Ala Met Leu Phe Phe
Ser Gly 165 170 175 Val Arg Thr Val Asp Pro Leu Trp Ala Leu Val Asp
Val Tyr Gly Leu 180 185 190 Thr Arg Gly Val Gln Leu Leu Asp Ser Glu
Leu Val Leu Pro Asp Cys 195 200 205 Leu Arg Pro Arg Ser Phe Thr Ala
Leu Arg Arg Pro Ser Leu Arg Cys 210 215 220 Glu Ala Asp Glu Ala Arg
Leu Ser Val Ser Leu Cys Asp Leu Asn Val 225 230 235 240 Pro Gly Ala
Asp Gly Asp Asp Gly Ala Pro Pro Ala Gly Cys Pro Ile 245 250 255 Pro
Gln Asn Ser Leu Asn Ser Gln His Ser Arg Ala Leu Pro Ala Gln 260 265
270 Leu Asp Gly Asp Leu Arg Phe His Ala Leu Arg Ala Gly Ala His Val
275 280 285 Arg Ile Leu Asp Glu Gln Thr Val Ala Arg Leu Glu His Gly
Arg Asp 290 295 300 Glu Arg Ala Leu Val Phe Thr Ser Arg Pro Val Ser
Val Ala Glu Thr 305 310 315 320 Ile Phe Ile Lys Val Thr Arg Ser Gly
Gly Gly Arg Ala Gly Ala Leu 325 330 335 Ser Phe Gly Val Thr Thr Cys
Asp Pro Gly Thr Leu Arg Pro Ala Asp 340 345 350 Leu Pro Phe Ser Pro
Glu Ala Leu Val Asp Arg Lys Glu Phe Trp Ala 355 360 365 Val Cys Arg
Val Pro Gly Pro Leu His Ser Gly Asp Ile Leu Gly Leu 370 375 380 Val
Val Asn Ala Asp Gly Glu Leu His Leu Ser His Asn Gly Ala Ala 385 390
395 400 Ala Gly Met Gln Leu Cys Val Asp Ala Ser Gln Pro Leu Trp Met
Leu 405 410 415 Phe Ser Leu His Gly Ala Ile Thr Gln Val Arg Ile Leu
Gly Ser Thr 420 425 430 Ile Met Thr Glu Arg Gly Gly Pro Ser Leu Pro
Cys Ser Pro Ala Ser 435 440 445 Thr Pro Thr Ser Pro Ser Ala Leu Gly
Ile Arg Leu Ser Asp Pro Leu 450 455 460 Leu Ser Thr Cys Gly Ser Gly
Pro Leu Gly Gly Ser Ala Gly Gly Thr 465 470 475 480 Ala Pro Asn Ser
Pro Val Ser Leu Pro Glu Ser Pro Val Thr Pro Gly 485 490 495 Leu Gly
Gln Trp Ser Asp Glu Cys Thr Ile Cys Tyr Glu His Ala Val 500 505 510
Asp Thr Val Ile Tyr Thr Cys Gly His Met Cys Leu Cys Tyr Ser Cys 515
520 525 Gly Leu Arg Leu Lys Lys Ala Leu His Ala Cys Cys Pro Ile Cys
Arg 530 535 540 Arg Pro Ile Lys Asp Ile Ile Lys Thr Tyr Arg Ser Ser
545 550 555 11 1035 DNA mouse 11 atgggtaaca acttctccag tgtctcctct
ctgcagcgag gaaacccgag ccgcgcgtcg 60 cggggccacc cccagaacct
caaagaatcc atcgggggct ccttcccggt gccctctcac 120 cgatgccatc
acaagcagaa gcattgcccg cctacgctgt caggtggggg gctcccggcc 180
acgccgctgc tcttccaccc ccacactaag ggctcccaga tcctcatgga cctcagccac
240 aaggccgtca agaggcaggc cagcttctgc aatgccatca ccttcagtaa
ccgcccggtg 300 ctcatctacg agcaagtcag gctgaagatc accaagaagc
aatgctgctg gagcggggcc 360 ctgcgacttg gcttcaccag caaggaccct
tcccgcatcc accccgactc gctgcccaag 420 tacgcctgcc ctgacctggt
gtctcagagt ggcttctggg ccaaagcatt gcctgaggag 480 tttgccaacg
agggcaacat cattgccttc tgggtggaca agaagggccg cgtcttctac 540
cggatcaatg agtcagctgc tatgcttttc ttcagtgggg tccggacggt ggacccgctc
600 tgggccctgg tggacgtcta cggcctcacg cggggtgtcc agctgctagg
ctccaccatc 660 atgactgaac ggggtggccc atctctcccc tgctcacctg
cttccactcc aacctcaccc 720 agtgccctgg gcatccgctt gtctgacccc
ttgctcagca cctgcggttc tgggccccta 780 ggtggctctg ctggagggac
agcccccaat tcacctgtga gcctgcccga gtcaccggtg 840 accccaggtc
tgggccagtg gagtgatgaa tgcaccattt gctatgaaca cgcagtggat 900
acagtcatct acacgtgtgg ccacatgtgc ctgtgctact cctgtggcct gcgcctcaag
960 aaggccctgc acgcctgctg ccccatctgc cgtcgcccca tcaaggacat
catcaagacc 1020 taccgcagct cgtag 1035 12 344 PRT mouse 12 Met Gly
Asn Asn Phe Ser Ser Val Ser Ser Leu Gln Arg Gly Asn Pro 1 5 10 15
Ser Arg Ala Ser Arg Gly His Pro Gln Asn Leu Lys Glu Ser Ile Gly 20
25 30 Gly Ser Phe Pro Val Pro Ser His Arg Cys His His Lys Gln Lys
His 35 40 45 Cys Pro Pro Thr Leu Ser Gly Gly Gly Leu Pro Ala Thr
Pro Leu Leu 50 55 60 Phe His Pro His Thr Lys Gly Ser Gln Ile Leu
Met Asp Leu Ser His 65 70 75 80 Lys Ala Val Lys Arg Gln Ala Ser Phe
Cys Asn Ala Ile Thr Phe Ser 85 90 95 Asn Arg Pro Val Leu Ile Tyr
Glu Gln Val Arg Leu Lys Ile Thr Lys 100 105 110 Lys Gln Cys Cys Trp
Ser Gly Ala Leu Arg Leu Gly Phe Thr Ser Lys 115 120 125 Asp Pro Ser
Arg Ile His Pro Asp Ser Leu Pro Lys Tyr Ala Cys Pro 130 135 140 Asp
Leu Val Ser Gln Ser Gly Phe Trp Ala Lys Ala Leu Pro Glu Glu 145 150
155 160 Phe Ala Asn Glu Gly Asn Ile Ile Ala Phe Trp Val Asp Lys Lys
Gly 165 170 175 Arg Val Phe Tyr Arg Ile Asn Glu Ser Ala Ala Met Leu
Phe Phe Ser 180 185 190 Gly Val Arg Thr Val Asp Pro Leu Trp Ala Leu
Val Asp Val Tyr Gly 195 200 205 Leu Thr Arg Gly Val Gln Leu Leu Gly
Ser Thr Ile Met Thr Glu Arg 210 215 220 Gly Gly Pro Ser Leu Pro Cys
Ser Pro Ala Ser Thr Pro Thr Ser Pro 225 230 235 240 Ser Ala Leu Gly
Ile Arg Leu Ser Asp Pro Leu Leu Ser Thr Cys Gly 245 250 255 Ser Gly
Pro Leu Gly Gly Ser Ala Gly Gly Thr Ala Pro Asn Ser Pro 260 265 270
Val Ser Leu Pro Glu Ser Pro Val Thr Pro Gly Leu Gly Gln Trp Ser 275
280 285 Asp Glu Cys Thr Ile Cys Tyr Glu His Ala Val Asp Thr Val Ile
Tyr 290 295 300 Thr Cys Gly His Met Cys Leu Cys Tyr Ser Cys Gly Leu
Arg Leu Lys 305 310 315 320 Lys Ala Leu His Ala Cys Cys Pro Ile Cys
Arg Arg Pro Ile Lys Asp 325 330 335 Ile Ile Lys Thr Tyr Arg Ser Ser
340 13 876 DNA mouse 13 atgggtaaca acttctccag tgtctcctct ctgcagcgag
gaaacccgag ccgcgcgtcg 60 cggggccacc cccagaacct caaagaatcc
atcgggggct ccttcccggt gccctctcac 120 cgatgccatc acaagcagaa
gcattgcccg cctacgctgt caggtggggg gctcccggcc 180 acgccgctgc
tcttccaccc ccacactaag ggctcccaga tcctcatgga cctcagccac 240
aaggccgtca agaggcaggc cagcttctgc aatgccatca ccttcagtaa ccgcccggtg
300 ctcatctacg agcaagtcag gctgaagatc accaagaagc aatgctgctg
gagcggggcc 360 ctgcgacttg gcttcaccag caaggaccct tcccgcatcc
accccgactc gctgcccaag 420 tacgcctgcc ctgacctggt gtctcagagt
ggcttctggg ccaaagcatt gcctgaggag 480 tttgccaacg agggcaacat
cattgccttc tgggtggaca agaagggccg cgtcttctac 540 cggatcaatg
agtcagctgc tatgcttttc ttcagtgggg tccggacggt ggacccgctc 600
tgggccctgg tggacgtcta cggcctcacg cggggtgtcc agctgctaga cagcgagctg
660 gtgctgcccg agtcaccggt gaccccaggt ctgggccagt ggagtgatga
atgcaccatt 720 tgctatgaac acgcagtgga tacagtcatc tacacgtgtg
gccacatgtg cctgtgctac 780 tcctgtggcc tgcgcctcaa gaaggccctg
cacgcctgct gccccatctg ccgtcgcccc 840 atcaaggaca tcatcaagac
ctaccgcagc tcgtag 876 14 291 PRT mouse 14 Met Gly Asn Asn Phe Ser
Ser Val Ser Ser Leu Gln Arg Gly Asn Pro 1 5 10 15 Ser Arg Ala Ser
Arg Gly His Pro Gln Asn Leu Lys Glu Ser Ile Gly 20 25 30 Gly Ser
Phe Pro Val Pro Ser His Arg Cys His His Lys Gln Lys His 35 40 45
Cys Pro Pro Thr Leu Ser Gly Gly Gly Leu Pro Ala Thr Pro Leu Leu 50
55 60 Phe His Pro His Thr Lys Gly Ser Gln Ile Leu Met Asp Leu Ser
His 65 70 75 80 Lys Ala Val Lys Arg Gln Ala Ser Phe Cys Asn Ala Ile
Thr Phe Ser 85 90 95 Asn Arg Pro Val Leu Ile Tyr Glu Gln Val Arg
Leu Lys Ile Thr Lys 100 105 110 Lys Gln Cys Cys Trp Ser Gly Ala Leu
Arg Leu Gly Phe Thr Ser Lys 115 120 125 Asp Pro Ser Arg Ile His Pro
Asp Ser Leu Pro Lys Tyr Ala Cys Pro 130 135 140 Asp Leu Val Ser Gln
Ser Gly Phe Trp Ala Lys Ala Leu Pro Glu Glu 145 150 155 160 Phe Ala
Asn Glu Gly Asn Ile Ile Ala Phe Trp Val Asp Lys Lys Gly 165 170 175
Arg Val Phe Tyr Arg Ile Asn Glu Ser Ala Ala Met Leu Phe Phe Ser 180
185 190 Gly Val Arg Thr Val Asp Pro Leu Trp Ala Leu Val Asp Val Tyr
Gly 195 200 205 Leu Thr Arg Gly Val Gln Leu Leu Asp Ser Glu Leu Val
Leu Pro Glu 210 215
220 Ser Pro Val Thr Pro Gly Leu Gly Gln Trp Ser Asp Glu Cys Thr Ile
225 230 235 240 Cys Tyr Glu His Ala Val Asp Thr Val Ile Tyr Thr Cys
Gly His Met 245 250 255 Cys Leu Cys Tyr Ser Cys Gly Leu Arg Leu Lys
Lys Ala Leu His Ala 260 265 270 Cys Cys Pro Ile Cys Arg Arg Pro Ile
Lys Asp Ile Ile Lys Thr Tyr 275 280 285 Arg Ser Ser 290 15 1725 DNA
rat 15 atgggtaaca acttctccag tgtctcctct ctgcagcgag gaaaccccag
ccgtgcgtcg 60 cggggccacc cccagaacct caaagactcc atcgggagct
ccttcccggt cccctctcac 120 cgatgccatc acaagcagaa gcattgcccg
cccgcgctgt caggtggggg gctcccggcc 180 acaccgctgc tcttccaccc
ccacactaag ggctcccaga tcctcatgga cctcagccac 240 aaggccgtca
agaggcaggc cagcttctgt aatgccatca ccttcagcaa ccgccccgtc 300
ctcatctacg agcaagtcag gctgaagatc accaagaagc agtgctgctg gagcggggcc
360 ctgcgacttg gcttcaccag caaggaccct tcccgcatcc accccgactc
actgcccaag 420 tacgcctgcc ctgacctggt gtcccagagt ggcttctggg
ccaaagcgtt gcctgaggag 480 tttgccaacg agggcaacat cattgccttc
tgggtggaca agaagggccg agtcttctac 540 cggatcaatg agtcggctgc
catgctgttc ttcagcgggg ttcgaacggc ggacccgctc 600 tgggccctgg
tggacgtcta tggcctcaca cggggtgtcc agctgctaga cagcgagctg 660
gtgctgcctg actgcctgag gccgcgctcc ttcaccgcgc tgcggcggcc gtcgctgcgg
720 tgcgaggcgg acgaggcgcg cctgtctgtg agcctgtgcg acctcaacgt
gccgggagcc 780 gacggcgagg acggcgcacc gcccgccggc tgcccgatcc
cgcagaactc gctcaactct 840 cagcacagcc gcgcgctgcc ggcgcagctc
gacggcgacc tgcgcttcca cgctctgcgc 900 gcccgcgcgc aagtccgcat
cctggacgag cagacggtgg cgcgcctaga gcacgggcgc 960 gacgagcgcg
cgctcgtctt caccagccgg ccggtgcgcg tggccgaaac catcttcatc 1020
aaggtcacgc gctcaggcgg tgcgcgacca gaagcgctgt ccttcggggt caccacgtgt
1080 gaccctggca cgctgcggcc cgccgacctg cccttcagcc ccgaggccct
ggtggaccgc 1140 aaggagttct gggcggtgtg tcgcgtgcct gggcctctgc
acagtggtga catcctgggc 1200 ctggtggtca atgcggacgg aaagctgcac
ctaattcaca acggcgcgcc ggccggcatg 1260 cagctatgcg tggacgcctc
gcagcccctc tggatgctct tcagcctgca cggcgccatc 1320 acgcaggtcc
gcatccttgg ctccaccatc atggctgaac ggggtggccc atctctcccc 1380
tgctcacctg cctccactcc aacctcgccc agtgccctgg gcagccgcct ctctgacccc
1440 ctgctcagca catgcggttc tgggcccctg ggtggctctg ttggcggaac
agcccccaac 1500 tcacctgtga gcctgcccga gtcaccagtg accccgggtc
tgggccagtg gagcgatgaa 1560 tgcaccattt gctatgaaca cgcagtggat
acagtcatct acacgtgtgg ccacatgtgc 1620 ctgtgctact cctgtggcct
gcgcctcaag aaggccctgc acgcctgctg ccccatctgc 1680 cgtcgcccca
tcaaggacat catcaagacc taccgcagct cctag 1725 16 574 PRT rat 16 Met
Gly Asn Asn Phe Ser Ser Val Ser Ser Leu Gln Arg Gly Asn Pro 1 5 10
15 Ser Arg Ala Ser Arg Gly His Pro Gln Asn Leu Lys Asp Ser Ile Gly
20 25 30 Ser Ser Phe Pro Val Pro Ser His Arg Cys His His Lys Gln
Lys His 35 40 45 Cys Pro Pro Ala Leu Ser Gly Gly Gly Leu Pro Ala
Thr Pro Leu Leu 50 55 60 Phe His Pro His Thr Lys Gly Ser Gln Ile
Leu Met Asp Leu Ser His 65 70 75 80 Lys Ala Val Lys Arg Gln Ala Ser
Phe Cys Asn Ala Ile Thr Phe Ser 85 90 95 Asn Arg Pro Val Leu Ile
Tyr Glu Gln Val Arg Leu Lys Ile Thr Lys 100 105 110 Lys Gln Cys Cys
Trp Ser Gly Ala Leu Arg Leu Gly Phe Thr Ser Lys 115 120 125 Asp Pro
Ser Arg Ile His Pro Asp Ser Leu Pro Lys Tyr Ala Cys Pro 130 135 140
Asp Leu Val Ser Gln Ser Gly Phe Trp Ala Lys Ala Leu Pro Glu Glu 145
150 155 160 Phe Ala Asn Glu Gly Asn Ile Ile Ala Phe Trp Val Asp Lys
Lys Gly 165 170 175 Arg Val Phe Tyr Arg Ile Asn Glu Ser Ala Ala Met
Leu Phe Phe Ser 180 185 190 Gly Val Arg Thr Ala Asp Pro Leu Trp Ala
Leu Val Asp Val Tyr Gly 195 200 205 Leu Thr Arg Gly Val Gln Leu Leu
Asp Ser Glu Leu Val Leu Pro Asp 210 215 220 Cys Leu Arg Pro Arg Ser
Phe Thr Ala Leu Arg Arg Pro Ser Leu Arg 225 230 235 240 Cys Glu Ala
Asp Glu Ala Arg Leu Ser Val Ser Leu Cys Asp Leu Asn 245 250 255 Val
Pro Gly Ala Asp Gly Glu Asp Gly Ala Pro Pro Ala Gly Cys Pro 260 265
270 Ile Pro Gln Asn Ser Leu Asn Ser Gln His Ser Arg Ala Leu Pro Ala
275 280 285 Gln Leu Asp Gly Asp Leu Arg Phe His Ala Leu Arg Ala Arg
Ala Gln 290 295 300 Val Arg Ile Leu Asp Glu Gln Thr Val Ala Arg Leu
Glu His Gly Arg 305 310 315 320 Asp Glu Arg Ala Leu Val Phe Thr Ser
Arg Pro Val Arg Val Ala Glu 325 330 335 Thr Ile Phe Ile Lys Val Thr
Arg Ser Gly Gly Ala Arg Pro Glu Ala 340 345 350 Leu Ser Phe Gly Val
Thr Thr Cys Asp Pro Gly Thr Leu Arg Pro Ala 355 360 365 Asp Leu Pro
Phe Ser Pro Glu Ala Leu Val Asp Arg Lys Glu Phe Trp 370 375 380 Ala
Val Cys Arg Val Pro Gly Pro Leu His Ser Gly Asp Ile Leu Gly 385 390
395 400 Leu Val Val Asn Ala Asp Gly Lys Leu His Leu Ile His Asn Gly
Ala 405 410 415 Pro Ala Gly Met Gln Leu Cys Val Asp Ala Ser Gln Pro
Leu Trp Met 420 425 430 Leu Phe Ser Leu His Gly Ala Ile Thr Gln Val
Arg Ile Leu Gly Ser 435 440 445 Thr Ile Met Ala Glu Arg Gly Gly Pro
Ser Leu Pro Cys Ser Pro Ala 450 455 460 Ser Thr Pro Thr Ser Pro Ser
Ala Leu Gly Ser Arg Leu Ser Asp Pro 465 470 475 480 Leu Leu Ser Thr
Cys Gly Ser Gly Pro Leu Gly Gly Ser Val Gly Gly 485 490 495 Thr Ala
Pro Asn Ser Pro Val Ser Leu Pro Glu Ser Pro Val Thr Pro 500 505 510
Gly Leu Gly Gln Trp Ser Asp Glu Cys Thr Ile Cys Tyr Glu His Ala 515
520 525 Val Asp Thr Val Ile Tyr Thr Cys Gly His Met Cys Leu Cys Tyr
Ser 530 535 540 Cys Gly Leu Arg Leu Lys Lys Ala Leu His Ala Cys Cys
Pro Ile Cys 545 550 555 560 Arg Arg Pro Ile Lys Asp Ile Ile Lys Thr
Tyr Arg Ser Ser 565 570 17 1035 DNA rat 17 atgggtaaca acttctccag
tgtctcctct ctgcagcgag gaaaccccag ccgtgcgtcg 60 cggggccacc
cccagaacct caaagactcc atcgggagct ccttcccggt cccctctcac 120
cgatgccatc acaagcagaa gcattgcccg cccgcgctgt caggtggggg gctcccggcc
180 acaccgctgc tcttccaccc ccacactaag ggctcccaga tcctcatgga
cctcagccac 240 aaggccgtca agaggcaggc cagcttctgt aatgccatca
ccttcagcaa ccgccccgtc 300 ctcatctacg agcaagtcag gctgaagatc
accaagaagc agtgctgctg gagcggggcc 360 ctgcgacttg gcttcaccag
caaggaccct tcccgcatcc accccgactc actgcccaag 420 tacgcctgcc
ctgacctggt gtcccagagt ggcttctggg ccaaagcgtt gcctgaggag 480
tttgccaacg agggcaacat cattgccttc tgggtggaca agaagggccg agtcttctac
540 cggatcaatg agtcggctgc catgctgttc ttcagcgggg ttcgaacggc
ggacccgctc 600 tgggccctgg tggacgtcta tggcctcaca cggggtgtcc
agctgctagg ctccaccatc 660 atggctgaac ggggtggccc atctctcccc
tgctcacctg cctccactcc aacctcgccc 720 agtgccctgg gcagccgcct
ctctgacccc ctgctcagca catgcggttc tgggcccctg 780 ggtggctctg
ttggcggaac agcccccaac tcacctgtga gcctgcccga gtcaccagtg 840
accccgggtc tgggccagtg gagcgatgaa tgcaccattt gctatgaaca cgcagtggat
900 acagtcatct acacgtgtgg ccacatgtgc ctgtgctact cctgtggcct
gcgcctcaag 960 aaggccctgc acgcctgctg ccccatctgc cgtcgcccca
tcaaggacat catcaagacc 1020 taccgcagct cctag 1035 18 344 PRT rat 18
Met Gly Asn Asn Phe Ser Ser Val Ser Ser Leu Gln Arg Gly Asn Pro 1 5
10 15 Ser Arg Ala Ser Arg Gly His Pro Gln Asn Leu Lys Asp Ser Ile
Gly 20 25 30 Ser Ser Phe Pro Val Pro Ser His Arg Cys His His Lys
Gln Lys His 35 40 45 Cys Pro Pro Ala Leu Ser Gly Gly Gly Leu Pro
Ala Thr Pro Leu Leu 50 55 60 Phe His Pro His Thr Lys Gly Ser Gln
Ile Leu Met Asp Leu Ser His 65 70 75 80 Lys Ala Val Lys Arg Gln Ala
Ser Phe Cys Asn Ala Ile Thr Phe Ser 85 90 95 Asn Arg Pro Val Leu
Ile Tyr Glu Gln Val Arg Leu Lys Ile Thr Lys 100 105 110 Lys Gln Cys
Cys Trp Ser Gly Ala Leu Arg Leu Gly Phe Thr Ser Lys 115 120 125 Asp
Pro Ser Arg Ile His Pro Asp Ser Leu Pro Lys Tyr Ala Cys Pro 130 135
140 Asp Leu Val Ser Gln Ser Gly Phe Trp Ala Lys Ala Leu Pro Glu Glu
145 150 155 160 Phe Ala Asn Glu Gly Asn Ile Ile Ala Phe Trp Val Asp
Lys Lys Gly 165 170 175 Arg Val Phe Tyr Arg Ile Asn Glu Ser Ala Ala
Met Leu Phe Phe Ser 180 185 190 Gly Val Arg Thr Ala Asp Pro Leu Trp
Ala Leu Val Asp Val Tyr Gly 195 200 205 Leu Thr Arg Gly Val Gln Leu
Leu Gly Ser Thr Ile Met Ala Glu Arg 210 215 220 Gly Gly Pro Ser Leu
Pro Cys Ser Pro Ala Ser Thr Pro Thr Ser Pro 225 230 235 240 Ser Ala
Leu Gly Ser Arg Leu Ser Asp Pro Leu Leu Ser Thr Cys Gly 245 250 255
Ser Gly Pro Leu Gly Gly Ser Val Gly Gly Thr Ala Pro Asn Ser Pro 260
265 270 Val Ser Leu Pro Glu Ser Pro Val Thr Pro Gly Leu Gly Gln Trp
Ser 275 280 285 Asp Glu Cys Thr Ile Cys Tyr Glu His Ala Val Asp Thr
Val Ile Tyr 290 295 300 Thr Cys Gly His Met Cys Leu Cys Tyr Ser Cys
Gly Leu Arg Leu Lys 305 310 315 320 Lys Ala Leu His Ala Cys Cys Pro
Ile Cys Arg Arg Pro Ile Lys Asp 325 330 335 Ile Ile Lys Thr Tyr Arg
Ser Ser 340 19 888 DNA rat 19 atgggtaaca acttctccag tgtctcctct
ctgcagcgag gaaaccccag ccgtgcgtcg 60 cggggccacc cccagaacct
caaagactcc atcgggagct ccttcccggt cccctctcac 120 cgatgccatc
acaagcagaa gcattgcccg cccgcgctgt caggtggggg gctcccggcc 180
acaccgctgc tcttccaccc ccacactaag ggctcccaga tcctcatgga cctcagccac
240 aaggccgtca agaggcaggc cagcttctgt aatgccatca ccttcagcaa
ccgccccgtc 300 ctcatctacg agcaagtcag gctgaagatc accaagaagc
agtgctgctg gagcggggcc 360 ctgcgacttg gcttcaccag caaggaccct
tcccgcatcc accccgactc actgcccaag 420 tacgcctgcc ctgacctggt
gtcccagagt ggcttctggg ccaaagcgtt gcctgaggag 480 tttgccaacg
agggcaacat cattgccttc tgggtggaca agaagggccg agtcttctac 540
cggatcaatg agtcggctgc catgctgttc ttcagcgggg ttcgaacggc ggacccgctc
600 tgggccctgg tggacgtcta tggcctcaca cggggtgtcc agctgctagg
aacagccccc 660 aactcacctg tgagcctgcc cgagtcacca gtgaccccgg
gtctgggcca gtggagcgat 720 gaatgcacca tttgctatga acacgcagtg
gatacagtca tctacacgtg tggccacatg 780 tgcctgtgct actcctgtgg
cctgcgcctc aagaaggccc tgcacgcctg ctgccccatc 840 tgccgtcgcc
ccatcaagga catcatcaag acctaccgca gctcctag 888 20 295 PRT rat 20 Met
Gly Asn Asn Phe Ser Ser Val Ser Ser Leu Gln Arg Gly Asn Pro 1 5 10
15 Ser Arg Ala Ser Arg Gly His Pro Gln Asn Leu Lys Asp Ser Ile Gly
20 25 30 Ser Ser Phe Pro Val Pro Ser His Arg Cys His His Lys Gln
Lys His 35 40 45 Cys Pro Pro Ala Leu Ser Gly Gly Gly Leu Pro Ala
Thr Pro Leu Leu 50 55 60 Phe His Pro His Thr Lys Gly Ser Gln Ile
Leu Met Asp Leu Ser His 65 70 75 80 Lys Ala Val Lys Arg Gln Ala Ser
Phe Cys Asn Ala Ile Thr Phe Ser 85 90 95 Asn Arg Pro Val Leu Ile
Tyr Glu Gln Val Arg Leu Lys Ile Thr Lys 100 105 110 Lys Gln Cys Cys
Trp Ser Gly Ala Leu Arg Leu Gly Phe Thr Ser Lys 115 120 125 Asp Pro
Ser Arg Ile His Pro Asp Ser Leu Pro Lys Tyr Ala Cys Pro 130 135 140
Asp Leu Val Ser Gln Ser Gly Phe Trp Ala Lys Ala Leu Pro Glu Glu 145
150 155 160 Phe Ala Asn Glu Gly Asn Ile Ile Ala Phe Trp Val Asp Lys
Lys Gly 165 170 175 Arg Val Phe Tyr Arg Ile Asn Glu Ser Ala Ala Met
Leu Phe Phe Ser 180 185 190 Gly Val Arg Thr Ala Asp Pro Leu Trp Ala
Leu Val Asp Val Tyr Gly 195 200 205 Leu Thr Arg Gly Val Gln Leu Leu
Gly Thr Ala Pro Asn Ser Pro Val 210 215 220 Ser Leu Pro Glu Ser Pro
Val Thr Pro Gly Leu Gly Gln Trp Ser Asp 225 230 235 240 Glu Cys Thr
Ile Cys Tyr Glu His Ala Val Asp Thr Val Ile Tyr Thr 245 250 255 Cys
Gly His Met Cys Leu Cys Tyr Ser Cys Gly Leu Arg Leu Lys Lys 260 265
270 Ala Leu His Ala Cys Cys Pro Ile Cys Arg Arg Pro Ile Lys Asp Ile
275 280 285 Ile Lys Thr Tyr Arg Ser Ser 290 295 21 1675 DNA Homo
sapien 21 atgggcaaca cggtgcaccg gaccctgcca gacccgagcc caccggcgcg
cctcctggcc 60 acccggccgt gctgcggccc cggccccgag cgacgcccgg
tcctgggcga ggcgccgcgc 120 ttccacgcgc aggccaaagg caagaacgtg
cggctggacg gccactcgcg ccgggccaca 180 cggcgcaaca gcttctgcaa
tggcgtcacg ttcacgcagc ggcccatccg gctgtacgag 240 caggtgcggc
tgcgcctggt ggccgtgcgc cctggctgga gcggcgcgct gcgcttcggc 300
ttcaccgcgc acgatccgtc gctcatgagc gcccaggaca tccccaagta cgcctgcccg
360 gacctggtca cgcggccggg ctactgggcc aaggcactgc ccgagaacct
ggcgctgcgc 420 gacacggtgc tggcctactg ggccgaccgc cacggccgcg
tgttctacag cgtgaacgac 480 ggcgagccgg tgctcttcca ctgcggcgtg
gccgtgggcg gcccgctctg ggcgctcatt 540 gatgtctacg gcatcaccga
cgaggtgcag cttctggaga gcgccttcgc tgacacgctg 600 acgcccgcgc
gcctcagcca ggcccgcttc agcgcctgcc tgccgcccag cagccacgac 660
gcggccaact tcgacaacaa cgagctcgag aacaaccagg tggtggccaa gctgggccac
720 ctggcgctgg gccgcgcccc gggcccaccg ccagccgacg ccgcggccgc
cgccattccg 780 tgcgggcccc gtgagcgccc gcggcccgcg tcgtcgccgg
cgctactgga ggccgacctg 840 cgcttccacg caacacgcgg gcccgacgtg
agcctgtcgg ccgaccgcaa agtggcctgc 900 gcaccgcggc ccgacggcgg
ccgcacgctg gtcttctccg agcgcccgct gcggcccggc 960 gagagcctct
tcgtggaggt gggccgtccg gggctggcgg cgcccggcgc gctggccttc 1020
ggcatcacgt cgtgcgaccc gggcgtgcta cggcccaacg agctgcccgc cgacccagac
1080 gcgctgctcg accgcaaaga gtactgggtg gtggcgcgcg ccgggcccgt
gccgagcggc 1140 ggcgacgcgc tcagcttcac gctgcggccc ggcggcgacg
tgctcctggg catcaacggg 1200 cgtccgcgcg gccgcctgct gtgcgtcgac
accacgcagg cgctctgggc cttcttcgcc 1260 gtgcgcggcg gcgtcgcggg
ccagctgcgt ctcctcggta ccctgcagtc cagccctgcg 1320 accacgactc
catcagggtc cctcagcggc tcccaggacg atagtgattc agatatgacc 1380
ttcagtgtca accagtcctc ctcggcatct gagtcatccc tggtgacggc ccccagctcc
1440 ccgctgagcc ccccggtgtc ccccgtgttc tccccaccgg agccggcagg
catcaagaat 1500 ggcgagtgca cggtgtgctt cgatggcgag gtggacacgg
tcatctacac gtgtggacac 1560 atgtgcctgt gccacagctg cggcctgcgg
ctcaagcgac aggcccgggc ctgctgcccc 1620 atctgccggc ggcccatcaa
ggacgtcatt aagatctaca ggccatagcc tagcc 1675 22 555 PRT Homo sapien
22 Met Gly Asn Thr Val His Arg Thr Leu Pro Asp Pro Ser Pro Pro Ala
1 5 10 15 Arg Leu Leu Ala Thr Arg Pro Cys Cys Gly Pro Gly Pro Glu
Arg Arg 20 25 30 Pro Val Leu Gly Glu Ala Pro Arg Phe His Ala Gln
Ala Lys Gly Lys 35 40 45 Asn Val Arg Leu Asp Gly His Ser Arg Arg
Ala Thr Arg Arg Asn Ser 50 55 60 Phe Cys Asn Gly Val Thr Phe Thr
Gln Arg Pro Ile Arg Leu Tyr Glu 65 70 75 80 Gln Val Arg Leu Arg Leu
Val Ala Val Arg Pro Gly Trp Ser Gly Ala 85 90 95 Leu Arg Phe Gly
Phe Thr Ala His Asp Pro Ser Leu Met Ser Ala Gln 100 105 110 Asp Ile
Pro Lys Tyr Ala Cys Pro Asp Leu Val Thr Arg Pro Gly Tyr 115 120 125
Trp Ala Lys Ala Leu Pro Glu Asn Leu Ala Leu Arg Asp Thr Val Leu 130
135 140 Ala Tyr Trp Ala Asp Arg His Gly Arg Val Phe Tyr Ser Val Asn
Asp 145 150 155 160 Gly Glu Pro Val Leu Phe His Cys Gly Val Ala Val
Gly Gly Pro Leu 165 170 175 Trp Ala Leu Ile Asp Val Tyr Gly Ile Thr
Asp Glu Val Gln Leu Leu 180 185 190 Glu Ser Ala Phe Ala Asp Thr Leu
Thr Pro Ala Arg Leu Ser Gln Ala 195 200 205 Arg Phe Ser Ala Cys Leu
Pro Pro Ser Ser His Asp Ala Ala Asn Phe 210 215 220 Asp Asn Asn Glu
Leu Glu Asn Asn Gln Val Val Ala Lys Leu Gly His 225 230 235 240 Leu
Ala Leu Gly Arg Ala Pro Gly Pro Pro Pro Ala Asp Ala Ala Ala 245 250
255 Ala Ala Ile Pro Cys Gly Pro Arg Glu Arg Pro Arg Pro Ala Ser Ser
260 265
270 Pro Ala Leu Leu Glu Ala Asp Leu Arg Phe His Ala Thr Arg Gly Pro
275 280 285 Asp Val Ser Leu Ser Ala Asp Arg Lys Val Ala Cys Ala Pro
Arg Pro 290 295 300 Asp Gly Gly Arg Thr Leu Val Phe Ser Glu Arg Pro
Leu Arg Pro Gly 305 310 315 320 Glu Ser Leu Phe Val Glu Val Gly Arg
Pro Gly Leu Ala Ala Pro Gly 325 330 335 Ala Leu Ala Phe Gly Ile Thr
Ser Cys Asp Pro Gly Val Leu Arg Pro 340 345 350 Asn Glu Leu Pro Ala
Asp Pro Asp Ala Leu Leu Asp Arg Lys Glu Tyr 355 360 365 Trp Val Val
Ala Arg Ala Gly Pro Val Pro Ser Gly Gly Asp Ala Leu 370 375 380 Ser
Phe Thr Leu Arg Pro Gly Gly Asp Val Leu Leu Gly Ile Asn Gly 385 390
395 400 Arg Pro Arg Gly Arg Leu Leu Cys Val Asp Thr Thr Gln Ala Leu
Trp 405 410 415 Ala Phe Phe Ala Val Arg Gly Gly Val Ala Gly Gln Leu
Arg Leu Leu 420 425 430 Gly Thr Leu Gln Ser Ser Pro Ala Thr Thr Thr
Pro Ser Gly Ser Leu 435 440 445 Ser Gly Ser Gln Asp Asp Ser Asp Ser
Asp Met Thr Phe Ser Val Asn 450 455 460 Gln Ser Ser Ser Ala Ser Glu
Ser Ser Leu Val Thr Ala Pro Ser Ser 465 470 475 480 Pro Leu Ser Pro
Pro Val Ser Pro Val Phe Ser Pro Pro Glu Pro Ala 485 490 495 Gly Ile
Lys Asn Gly Glu Cys Thr Val Cys Phe Asp Gly Glu Val Asp 500 505 510
Thr Val Ile Tyr Thr Cys Gly His Met Cys Leu Cys His Ser Cys Gly 515
520 525 Leu Arg Leu Lys Arg Gln Ala Arg Ala Cys Cys Pro Ile Cys Arg
Arg 530 535 540 Pro Ile Lys Asp Val Ile Lys Ile Tyr Arg Pro 545 550
555 23 1129 DNA Homo sapien 23 atgggcaaca cggtgcaccg gaccctgcca
gagagcgcct tcgctgacac gctgacgccc 60 gcgcgcctca gccaggcccg
cttcagcgcc tgcctgccgc ccagcagcca cgacgcggcc 120 aacttcgaca
acaacgagct cgagaacaac caggtggtgg ccaagctggg ccacctggcg 180
ctgggccgcg ccccgggccc accgccagcc gacgccgcgg ccgccgccat tccgtgcggg
240 ccccgtgagc gcccgcggcc cgcgtcgtcg ccggcgctac tggaggccga
cctgcgcttc 300 cacgcaacac gcgggcccga cgtgagcctg tcggccgacc
gcaaagtggc ctgcgcaccg 360 cggcccgacg gcggccgcac gctggtcttc
tccgagcgcc cgctgcggcc cggcgagagc 420 ctcttcgtgg aggtgggccg
tccggggctg gcggcgcccg gcgcgctggc cttcggcatc 480 acgtcgtgcg
acccgggcgt gctacggccc aacgagctgc ccgccgaccc agacgcgctg 540
ctcgaccgca aagagtactg ggtggtggcg cgcgccgggc ccgtgccgag cggcggcgac
600 gcgctcagct tcacgctgcg gcccggcggc gacgtgctcc tgggcatcaa
cgggcgtccg 660 cgcggccgcc tgctgtgcgt cgacaccacg caggcgctct
gggccttctt cgccgtgcgc 720 ggcggcgtcg cgggccagct gcgtctcctc
ggtaccctgc agtccagccc tgcgaccacg 780 actccatcag ggtccctcag
cggctcccag gacgatagtg attcagatat gaccttcagt 840 gtcaaccagt
cctcctcggc atctgagtca tccctggtga cggcccccag ctccccgctg 900
agccccccgg tgtcccccgt gttctcccca ccggagccgg caggcatcaa gaatggcgag
960 tgcacggtgt gcttcgatgg cgaggtggac acggtcatct acacgtgtgg
acacatgtgc 1020 ctgtgccaca gctgcggcct gcggctcaag cgacaggccc
gggcctgctg ccccatctgc 1080 cggcggccca tcaaggacgt cattaagatc
tacaggccat agcctagcc 1129 24 373 PRT Homo sapien 24 Met Gly Asn Thr
Val His Arg Thr Leu Pro Glu Ser Ala Phe Ala Asp 1 5 10 15 Thr Leu
Thr Pro Ala Arg Leu Ser Gln Ala Arg Phe Ser Ala Cys Leu 20 25 30
Pro Pro Ser Ser His Asp Ala Ala Asn Phe Asp Asn Asn Glu Leu Glu 35
40 45 Asn Asn Gln Val Val Ala Lys Leu Gly His Leu Ala Leu Gly Arg
Ala 50 55 60 Pro Gly Pro Pro Pro Ala Asp Ala Ala Ala Ala Ala Ile
Pro Cys Gly 65 70 75 80 Pro Arg Glu Arg Pro Arg Pro Ala Ser Ser Pro
Ala Leu Leu Glu Ala 85 90 95 Asp Leu Arg Phe His Ala Thr Arg Gly
Pro Asp Val Ser Leu Ser Ala 100 105 110 Asp Arg Lys Val Ala Cys Ala
Pro Arg Pro Asp Gly Gly Arg Thr Leu 115 120 125 Val Phe Ser Glu Arg
Pro Leu Arg Pro Gly Glu Ser Leu Phe Val Glu 130 135 140 Val Gly Arg
Pro Gly Leu Ala Ala Pro Gly Ala Leu Ala Phe Gly Ile 145 150 155 160
Thr Ser Cys Asp Pro Gly Val Leu Arg Pro Asn Glu Leu Pro Ala Asp 165
170 175 Pro Asp Ala Leu Leu Asp Arg Lys Glu Tyr Trp Val Val Ala Arg
Ala 180 185 190 Gly Pro Val Pro Ser Gly Gly Asp Ala Leu Ser Phe Thr
Leu Arg Pro 195 200 205 Gly Gly Asp Val Leu Leu Gly Ile Asn Gly Arg
Pro Arg Gly Arg Leu 210 215 220 Leu Cys Val Asp Thr Thr Gln Ala Leu
Trp Ala Phe Phe Ala Val Arg 225 230 235 240 Gly Gly Val Ala Gly Gln
Leu Arg Leu Leu Gly Thr Leu Gln Ser Ser 245 250 255 Pro Ala Thr Thr
Thr Pro Ser Gly Ser Leu Ser Gly Ser Gln Asp Asp 260 265 270 Ser Asp
Ser Asp Met Thr Phe Ser Val Asn Gln Ser Ser Ser Ala Ser 275 280 285
Glu Ser Ser Leu Val Thr Ala Pro Ser Ser Pro Leu Ser Pro Pro Val 290
295 300 Ser Pro Val Phe Ser Pro Pro Glu Pro Ala Gly Ile Lys Asn Gly
Glu 305 310 315 320 Cys Thr Val Cys Phe Asp Gly Glu Val Asp Thr Val
Ile Tyr Thr Cys 325 330 335 Gly His Met Cys Leu Cys His Ser Cys Gly
Leu Arg Leu Lys Arg Gln 340 345 350 Ala Arg Ala Cys Cys Pro Ile Cys
Arg Arg Pro Ile Lys Asp Val Ile 355 360 365 Lys Ile Tyr Arg Pro 370
25 955 DNA Homo sapien 25 atgggcaaca cggtgcaccg gaccctgcca
gacccgagcc caccggcgcg cctcctggcc 60 acccggccgt gctgcggccc
cggccccgag cgacgcccgg tcctgggcga ggcgccgcgc 120 ttccacgcgc
aggccaaagg caagaacgtg cggctggacg gccactcgcg ccgggccaca 180
cggcgcaaca gcttctgcaa tggcgtcacg ttcacgcagc ggcccatccg gctgtacgag
240 caggtgcggc tgcgcctggt ggccgtgcgc cctggctgga gcggcgcgct
gcgcttcggc 300 ttcaccgcgc acgatccgtc gctcatgagc gcccaggaca
tccccaagta cgcctgcccg 360 gacctggtca cgcggccggg ctactgggcc
aaggcactgc ccgagaacct ggcgctgcgc 420 gacacggtgc tggcctactg
ggccgaccgc cacggccgcg tgttctacag cgtgaacgac 480 ggcgagccgg
tgctcttcca ctgcggcgtg gccgtgggcg gcccgctctg ggcgctcatt 540
gatgtctacg gcatcaccga cgaggtgcag cttctgggta ccctgcagtc cagccctgcg
600 accacgactc catcagggtc cctcagcggc tcccaggacg atagtgattc
agatatgacc 660 ttcagtgtca accagtcctc ctcggcatct gagtcatccc
tggtgacggc ccccagctcc 720 ccgctgagcc ccccggtgtc ccccgtgttc
tccccaccgg agccggcagg catcaagaat 780 ggcgagtgca cggtgtgctt
cgatggcgag gtggacacgg tcatctacac gtgtggacac 840 atgtgcctgt
gccacagctg cggcctgcgg ctcaagcgac aggcccgggc ctgctgcccc 900
atctgccggc ggcccatcaa ggacgtcatt aagatctaca ggccatagcc tagcc 955 26
315 PRT Homo sapien 26 Met Gly Asn Thr Val His Arg Thr Leu Pro Asp
Pro Ser Pro Pro Ala 1 5 10 15 Arg Leu Leu Ala Thr Arg Pro Cys Cys
Gly Pro Gly Pro Glu Arg Arg 20 25 30 Pro Val Leu Gly Glu Ala Pro
Arg Phe His Ala Gln Ala Lys Gly Lys 35 40 45 Asn Val Arg Leu Asp
Gly His Ser Arg Arg Ala Thr Arg Arg Asn Ser 50 55 60 Phe Cys Asn
Gly Val Thr Phe Thr Gln Arg Pro Ile Arg Leu Tyr Glu 65 70 75 80 Gln
Val Arg Leu Arg Leu Val Ala Val Arg Pro Gly Trp Ser Gly Ala 85 90
95 Leu Arg Phe Gly Phe Thr Ala His Asp Pro Ser Leu Met Ser Ala Gln
100 105 110 Asp Ile Pro Lys Tyr Ala Cys Pro Asp Leu Val Thr Arg Pro
Gly Tyr 115 120 125 Trp Ala Lys Ala Leu Pro Glu Asn Leu Ala Leu Arg
Asp Thr Val Leu 130 135 140 Ala Tyr Trp Ala Asp Arg His Gly Arg Val
Phe Tyr Ser Val Asn Asp 145 150 155 160 Gly Glu Pro Val Leu Phe His
Cys Gly Val Ala Val Gly Gly Pro Leu 165 170 175 Trp Ala Leu Ile Asp
Val Tyr Gly Ile Thr Asp Glu Val Gln Leu Leu 180 185 190 Gly Thr Leu
Gln Ser Ser Pro Ala Thr Thr Thr Pro Ser Gly Ser Leu 195 200 205 Ser
Gly Ser Gln Asp Asp Ser Asp Ser Asp Met Thr Phe Ser Val Asn 210 215
220 Gln Ser Ser Ser Ala Ser Glu Ser Ser Leu Val Thr Ala Pro Ser Ser
225 230 235 240 Pro Leu Ser Pro Pro Val Ser Pro Val Phe Ser Pro Pro
Glu Pro Ala 245 250 255 Gly Ile Lys Asn Gly Glu Cys Thr Val Cys Phe
Asp Gly Glu Val Asp 260 265 270 Thr Val Ile Tyr Thr Cys Gly His Met
Cys Leu Cys His Ser Cys Gly 275 280 285 Leu Arg Leu Lys Arg Gln Ala
Arg Ala Cys Cys Pro Ile Cys Arg Arg 290 295 300 Pro Ile Lys Asp Val
Ile Lys Ile Tyr Arg Pro 305 310 315 27 1641 DNA Rat 27 atgggcaaca
cggtgcaccg gaccctgcca gactccagcc ctcctgcacg cctcctggcc 60
acccggcctt gctacggccc gggcccggag aggcgagcgg tcctgggcga ggcgccacgc
120 ttccacgcgc aggccaaggg caagaacgtg cgtctagacg gtcactcgcg
cagggccacg 180 cgacggaaca gcttctgcaa cggagtcacc ttcacgcagc
ggcccattcg cctgtacgag 240 caggtgcggt tgcgcctggt ggctgtgcgt
cctggctgga gcggcgcgct gcgcttcggc 300 ttcactgcgc acgacccgtc
gctcatgagc gcacaggata tccccaagta cgcctgcccc 360 gacctggtca
cacgacctgg atactgggcc aaggcgctgc cggagaacct ggcgctgcgg 420
gacacggtgc tggcctactg ggctgatcgt cacggtcgcg tcttctatag tgtctatgat
480 ggcgaaccag tgctgttcca ctgcggcgtg gccgtgggaa gcccactctg
ggcactcatc 540 gacgtctatg gcatcacgga cgaggtgcag ctgctggaaa
gcacctgcgc agacacgctg 600 accccgctgc gcctgggcca ggcccgcctc
agcgcctgcc cgcctccggg cagccacgat 660 gctgccaact tcgataataa
cgagctggag aataaccagg tggtagccaa gctgggtcac 720 ttggctctcg
gccgtccgga cgccgccgtc ccgtgcgtgg cccgcgaacg cgcgaggccc 780
gcttcttcac ccgcgttgct ggacgctgag ctgcgtttcc acgccacgcg cggccccgac
840 gtgagcctgt ttgcggaccg caggttagct tgcgcgcccc gccccgacgg
cggccgcacg 900 tttgtgttct ccgagcggcc gctgcggccc ggggagagcc
tgtgcgtgga agtggggcgc 960 ccggggctgg cggcgcccgc agctgtggcc
ttcggcatca cgtcctgcga tcctggcgcg 1020 ctgcggccat ccgagctgcc
cgccgatccc gctgcgctgc tggaccgcaa ggaatactgg 1080 gtggtggcgc
gcgccgggcc cgtgcccagc ggaggcgacg cactcagctt cacgctgcga 1140
ccgggcggcg acgtcctgct ggcggtgaac gggcgcccgc ggggacgctt gctgtgcgtg
1200 gacacctcgc aggcgctctg ggccttcttc gctgtgcgcg gtggtgtggc
gggtcagctg 1260 cgtctcctgg gcaccgtaca gtccggtcct gaggccacaa
ctccatcagg gtccttcagt 1320 ggctctcagg atgacagcga ttcggacatg
accttcgggg tcaaccagtc gtcatcagca 1380 tcagaatcgt ctctggtgac
agcccccagc tccccactga gtcccccagt gtccccggcc 1440 ttctctgcac
ccgagccggc cggcagcagg aatggagagt gcacggtgtg cttcgacagc 1500
gaggtggaca cggtcatcta cacgtgtgga cacatgtgcc tgtgccacag ctgccgcctg
1560 cggctccgaa agcaggcacg ggcctgctgt cctatctgcc gtcggcccat
caaggatgtt 1620 atcaagatct ataggccgta g 1641 28 546 PRT Rat 28 Met
Gly Asn Thr Val His Arg Thr Leu Pro Asp Ser Ser Pro Pro Ala 1 5 10
15 Arg Leu Leu Ala Thr Arg Pro Cys Tyr Gly Pro Gly Pro Glu Arg Arg
20 25 30 Ala Val Leu Gly Glu Ala Pro Arg Phe His Ala Gln Ala Lys
Gly Lys 35 40 45 Asn Val Arg Leu Asp Gly His Ser Arg Arg Ala Thr
Arg Arg Asn Ser 50 55 60 Phe Cys Asn Gly Val Thr Phe Thr Gln Arg
Pro Ile Arg Leu Tyr Glu 65 70 75 80 Gln Val Arg Leu Arg Leu Val Ala
Val Arg Pro Gly Trp Ser Gly Ala 85 90 95 Leu Arg Phe Gly Phe Thr
Ala His Asp Pro Ser Leu Met Ser Ala Gln 100 105 110 Asp Ile Pro Lys
Tyr Ala Cys Pro Asp Leu Val Thr Arg Pro Gly Tyr 115 120 125 Trp Ala
Lys Ala Leu Pro Glu Asn Leu Ala Leu Arg Asp Thr Val Leu 130 135 140
Ala Tyr Trp Ala Asp Arg His Gly Arg Val Phe Tyr Ser Val Tyr Asp 145
150 155 160 Gly Glu Pro Val Leu Phe His Cys Gly Val Ala Val Gly Ser
Pro Leu 165 170 175 Trp Ala Leu Ile Asp Val Tyr Gly Ile Thr Asp Glu
Val Gln Leu Leu 180 185 190 Glu Ser Thr Cys Ala Asp Thr Leu Thr Pro
Leu Arg Leu Gly Gln Ala 195 200 205 Arg Leu Ser Ala Cys Pro Pro Pro
Gly Ser His Asp Ala Ala Asn Phe 210 215 220 Asp Asn Asn Glu Leu Glu
Asn Asn Gln Val Val Ala Lys Leu Gly His 225 230 235 240 Leu Ala Leu
Gly Arg Pro Asp Ala Ala Val Pro Cys Val Ala Arg Glu 245 250 255 Arg
Ala Arg Pro Ala Ser Ser Pro Ala Leu Leu Asp Ala Glu Leu Arg 260 265
270 Phe His Ala Thr Arg Gly Pro Asp Val Ser Leu Phe Ala Asp Arg Arg
275 280 285 Leu Ala Cys Ala Pro Arg Pro Asp Gly Gly Arg Thr Phe Val
Phe Ser 290 295 300 Glu Arg Pro Leu Arg Pro Gly Glu Ser Leu Cys Val
Glu Val Gly Arg 305 310 315 320 Pro Gly Leu Ala Ala Pro Ala Ala Val
Ala Phe Gly Ile Thr Ser Cys 325 330 335 Asp Pro Gly Ala Leu Arg Pro
Ser Glu Leu Pro Ala Asp Pro Ala Ala 340 345 350 Leu Leu Asp Arg Lys
Glu Tyr Trp Val Val Ala Arg Ala Gly Pro Val 355 360 365 Pro Ser Gly
Gly Asp Ala Leu Ser Phe Thr Leu Arg Pro Gly Gly Asp 370 375 380 Val
Leu Leu Ala Val Asn Gly Arg Pro Arg Gly Arg Leu Leu Cys Val 385 390
395 400 Asp Thr Ser Gln Ala Leu Trp Ala Phe Phe Ala Val Arg Gly Gly
Val 405 410 415 Ala Gly Gln Leu Arg Leu Leu Gly Thr Val Gln Ser Gly
Pro Glu Ala 420 425 430 Thr Thr Pro Ser Gly Ser Phe Ser Gly Ser Gln
Asp Asp Ser Asp Ser 435 440 445 Asp Met Thr Phe Gly Val Asn Gln Ser
Ser Ser Ala Ser Glu Ser Ser 450 455 460 Leu Val Thr Ala Pro Ser Ser
Pro Leu Ser Pro Pro Val Ser Pro Ala 465 470 475 480 Phe Ser Ala Pro
Glu Pro Ala Gly Ser Arg Asn Gly Glu Cys Thr Val 485 490 495 Cys Phe
Asp Ser Glu Val Asp Thr Val Ile Tyr Thr Cys Gly His Met 500 505 510
Cys Leu Cys His Ser Cys Arg Leu Arg Leu Arg Lys Gln Ala Arg Ala 515
520 525 Cys Cys Pro Ile Cys Arg Arg Pro Ile Lys Asp Val Ile Lys Ile
Tyr 530 535 540 Arg Pro 545 29 789 DNA Homo sapien 29 atgggtgccc
agctctgctt tgaggccaac gccaaggcgc cccgagaggc acttcgcttc 60
catgccgagg ccaagggcgc acaggtgcgt ctggacacgc gtggctgcat cgcgcacagg
120 cgcaccacgt tccacgacgg catcgtgttc agccagcggc cggtgcgcct
gggcgagcgt 180 gtggcgctgc gagtgctgcg ggaggagagc ggctggtgcg
gcggcctccg cgtgggcttc 240 acgcgcctgg accccgcgtg cgtgtccgtg
cccagcctgc cgcccttcct gtgccccgac 300 ctggaggagc agagcccgac
gtgggcggcc gtgctgcctg agggctgcgc gctcactggg 360 gacttggtcc
gcttctgggt ggaccgccgc ggctgcctct tcgccaaggt caacgccggc 420
tgccggctcc tgctgcgtga gggcgtgccc gtcggcgccc cgctctgggc cgtgatggac
480 gtgtatggga ccactaaggc catcgagctg ctggatccca cagccagccg
gctcccaaca 540 cccatgccat gggacctcag caacaaggct gtgcctgagc
ccaaagccac accaggagag 600 gagtgtgcca tctgcttcta tcacgctgcc
aacacccgcc ttgtgccctg cggccacaca 660 tacttctgca gatactgtgc
ctggcgggtc ttcagcgata cggccaagtg ccctgtgtgc 720 cgctggcaga
tagaggcggt agcccctgcg cagggccctc ctgctctgag ggttgaggaa 780
ggctcatga 789 30 262 PRT Homo sapien 30 Met Gly Ala Gln Leu Cys Phe
Glu Ala Asn Ala Lys Ala Pro Arg Glu 1 5 10 15 Ala Leu Arg Phe His
Ala Glu Ala Lys Gly Ala Gln Val Arg Leu Asp 20 25 30 Thr Arg Gly
Cys Ile Ala His Arg Arg Thr Thr Phe His Asp Gly Ile 35 40 45 Val
Phe Ser Gln Arg Pro Val Arg Leu Gly Glu Arg Val Ala Leu Arg 50 55
60 Val Leu Arg Glu Glu Ser Gly Trp Cys Gly Gly Leu Arg Val Gly Phe
65 70 75 80 Thr Arg Leu Asp Pro Ala Cys Val Ser Val Pro Ser Leu Pro
Pro Phe 85 90 95 Leu Cys Pro Asp Leu Glu Glu Gln Ser Pro Thr Trp
Ala Ala Val Leu 100 105 110 Pro Glu Gly Cys Ala Leu Thr Gly Asp Leu
Val Arg Phe Trp Val Asp 115 120 125 Arg Arg Gly Cys Leu Phe Ala Lys
Val Asn Ala Gly Cys Arg Leu Leu 130 135 140 Leu Arg Glu Gly Val Pro
Val Gly Ala Pro Leu Trp Ala Val Met Asp 145 150
155 160 Val Tyr Gly Thr Thr Lys Ala Ile Glu Leu Leu Asp Pro Thr Ala
Ser 165 170 175 Arg Leu Pro Thr Pro Met Pro Trp Asp Leu Ser Asn Lys
Ala Val Pro 180 185 190 Glu Pro Lys Ala Thr Pro Gly Glu Glu Cys Ala
Ile Cys Phe Tyr His 195 200 205 Ala Ala Asn Thr Arg Leu Val Pro Cys
Gly His Thr Tyr Phe Cys Arg 210 215 220 Tyr Cys Ala Trp Arg Val Phe
Ser Asp Thr Ala Lys Cys Pro Val Cys 225 230 235 240 Arg Trp Gln Ile
Glu Ala Val Ala Pro Ala Gln Gly Pro Pro Ala Leu 245 250 255 Arg Val
Glu Glu Gly Ser 260 31 765 DNA mouse 31 atgggttctc tcctcagccc
tgaggccaat gccgaggtgc cccgcgaggc ccttagtttc 60 cacgggaacg
ccacgggcgc acaggtgcat ctggacgatc agcggagcac agcgcgcagg 120
cgctcgacgt tccacgatgg tatcgtgttc agccagaggc cggtctggcc gggtgagcgt
180 gttgctctgc gcgtcctgcg acatgaagaa ggctggtgcg gtggcctccg
cgtgggcttc 240 acgcgcctgg accctgcgca agtggccgcg tcctgcctgc
cacccttcgt gtgcccggac 300 ctggaggagc agagtcccac gtgggcagcg
ttgcttccag agggcttcgt tcgtgcgggg 360 aatgtggtct gcttctgggt
gaaccgtaga gggtggctct tcgccaaggt caacgctggc 420 cgccccctct
tgctgcgcaa agacgtgctg gtccagggcg ccccgctctg ggcggtgatg 480
gatgtgtacg ggaccacgaa agccattgag cttctggatc ccaaagccaa cgcctggatt
540 cgtagtggtg agcctgtgcc agagtctgaa gtcatatcag gagaggagtg
tgtcatctgc 600 ttccacaaca ctgccaacac ccgcctcatg ccctgtggcc
actcacactt ctgtggctcc 660 tgtgcctggc acatcttcaa agacacggcc
aggtgcccca tatgtcgctg gcagatcgag 720 gaggtggctg tagtgtcttc
actgaaggct gaggaaggct cctga 765 32 254 PRT mouse 32 Met Gly Ser Leu
Leu Ser Pro Glu Ala Asn Ala Glu Val Pro Arg Glu 1 5 10 15 Ala Leu
Ser Phe His Gly Asn Ala Thr Gly Ala Gln Val His Leu Asp 20 25 30
Asp Gln Arg Ser Thr Ala Arg Arg Arg Ser Thr Phe His Asp Gly Ile 35
40 45 Val Phe Ser Gln Arg Pro Val Trp Pro Gly Glu Arg Val Ala Leu
Arg 50 55 60 Val Leu Arg His Glu Glu Gly Trp Cys Gly Gly Leu Arg
Val Gly Phe 65 70 75 80 Thr Arg Leu Asp Pro Ala Gln Val Ala Ala Ser
Cys Leu Pro Pro Phe 85 90 95 Val Cys Pro Asp Leu Glu Glu Gln Ser
Pro Thr Trp Ala Ala Leu Leu 100 105 110 Pro Glu Gly Phe Val Arg Ala
Gly Asn Val Val Cys Phe Trp Val Asn 115 120 125 Arg Arg Gly Trp Leu
Phe Ala Lys Val Asn Ala Gly Arg Pro Leu Leu 130 135 140 Leu Arg Lys
Asp Val Leu Val Gln Gly Ala Pro Leu Trp Ala Val Met 145 150 155 160
Asp Val Tyr Gly Thr Thr Lys Ala Ile Glu Leu Leu Asp Pro Lys Ala 165
170 175 Asn Ala Trp Ile Arg Ser Gly Glu Pro Val Pro Glu Ser Glu Val
Ile 180 185 190 Ser Gly Glu Glu Cys Val Ile Cys Phe His Asn Thr Ala
Asn Thr Arg 195 200 205 Leu Met Pro Cys Gly His Ser His Phe Cys Gly
Ser Cys Ala Trp His 210 215 220 Ile Phe Lys Asp Thr Ala Arg Cys Pro
Ile Cys Arg Trp Gln Ile Glu 225 230 235 240 Glu Val Ala Val Val Ser
Ser Leu Lys Ala Glu Glu Gly Ser 245 250 33 250 DNA Homo sapien 33
cgcttccatc gggtgcacgg tgccaacatc cgcgtggacc cctctgggac gcgggccaca
60 cgcgtggaga gcttcgccca cggcgtgtgc ttcagccgcg agccgctggc
cccgggccag 120 gtcttcctgg tcgagatcga ggagaaagag ctgggctggt
gcggacatct gcgtctcggt 180 ctgaccgcgc tggaccccgc cagtctggcc
cccgttcccg agttttctct gcccgatctg 240 gtcaacctgg 250 34 83 PRT Homo
sapien 34 Arg Phe His Arg Val His Gly Ala Asn Ile Arg Val Asp Pro
Ser Gly 1 5 10 15 Thr Arg Ala Thr Arg Val Glu Ser Phe Ala His Gly
Val Cys Phe Ser 20 25 30 Arg Glu Pro Leu Ala Pro Gly Gln Val Phe
Leu Val Glu Ile Glu Glu 35 40 45 Lys Glu Leu Gly Trp Cys Gly His
Leu Arg Leu Gly Leu Thr Ala Leu 50 55 60 Asp Pro Ala Ser Leu Ala
Pro Val Pro Glu Phe Ser Leu Pro Asp Leu 65 70 75 80 Val Asn Leu 35
1743 DNA rat 35 atggcggatt tcgaggagtt gaggaatatg gtttctagtt
ttagggtttc tgaattacaa 60 gtgttactgg gctttgctgg acggaataaa
agtgggcgca agcatgacct cctgatgagg 120 gcgttgcatt tactgaagag
tggctgcagc cctgcggttc agattaaaat tcgagaatta 180 tacagacgcc
gatacccacg gacacttgaa ggactttctg atctatccac aatcaaatct 240
tcagttttca gtttggatgg tagctcatca ccagtagagc ctgacttggc cgtggctggg
300 atccactcgt tgccttctac ttccattgca cctcattcac cgtcatctcc
tgtcgcttct 360 gtgctgcttc aagacactaa gcccacgttt gagatgcagc
aaccatctcc tcccattcct 420 cctgtccatc ctgacgtgca gttaaaaacg
ctgcccttct atgacgtcct tgatgttctc 480 atcaagccca caagtttagt
tcaaagcagt attcagcggt ttcaagagaa gttttttatt 540 tttgctttga
caccccagca agttagagag atatgcattt caagggattt tttgccaggt 600
ggcaggagag actacacagt ccaagtccag ctgcgacttt gcttggcaga gaccagttgc
660 cctcaagaag ataactatcc caatagtttg tgtataaaag taaatgggaa
actctttcct 720 ttgcctggct atgcaccacc acctaaaaat gggatcgaac
agaagcgtcc tggacgcccc 780 ctgaatatta catctttagt gagattgtct
tcagctgtgc caaatcagat ttctatttct 840 tgggcatctg aaattggaaa
gaattactcc atgtctgtgt atcttgtacg acagcttaca 900 tcagccatgt
tattacagag attaaaaatg aaaggtatta gaaatcctga tcattccaaa 960
gcactcatta aagaaaaact tactgcagat cctgatagtg aaattgctac aactagtctt
1020 cgagtgtcct tgatgtgccc tttaggaaaa atgaggctga caatcccgtg
ccgcgcagtg 1080 acgtgtacac atctgcagtg ctttgatgct gccctgtatc
ttcagatgaa tgagaagaag 1140 cccacctgga tttgtcctgt ttgtgacaaa
aaggctgcct atgagagtct gatactagat 1200 gggcttttta tggaaattct
caatgactgt tctgatgtgg atgagatcaa attccaggaa 1260 gatggttcct
ggtgccccat gagacctaag aaagaagcta tgaaagtaac cagccagccc 1320
tgtacaaaag tagaaagttc aagtgtcttt agtaaacctt gttcagtgac tgtagccagt
1380 gatgcaagca agaagaagat tgatgttatt gatctaacaa tagagagctc
ttctgatgaa 1440 gaggaagacc ctcccgccaa aaggaaatgc atctttatgt
cagaaacaca aagcagtcca 1500 accaaagggg ttctcatgta tcagccatct
tctgtaaggg tgcccagtgt gacttcagtt 1560 gatcctgctg ctattccacc
ttcattaaca gactactcag taccattcca ccacacgcca 1620 gtgtcgagca
tgtcatcaga tttgccaggt ttggattttc tttcccttat tccagttgat 1680
ccccagtctc acctcaccct taacagcaag cagtacgtct gtcaccacca ccagccccca
1740 tga 1743 36 580 PRT rat 36 Met Ala Asp Phe Glu Glu Leu Arg Asn
Met Val Ser Ser Phe Arg Val 1 5 10 15 Ser Glu Leu Gln Val Leu Leu
Gly Phe Ala Gly Arg Asn Lys Ser Gly 20 25 30 Arg Lys His Asp Leu
Leu Met Arg Ala Leu His Leu Leu Lys Ser Gly 35 40 45 Cys Ser Pro
Ala Val Gln Ile Lys Ile Arg Glu Leu Tyr Arg Arg Arg 50 55 60 Tyr
Pro Arg Thr Leu Glu Gly Leu Ser Asp Leu Ser Thr Ile Lys Ser 65 70
75 80 Ser Val Phe Ser Leu Asp Gly Ser Ser Ser Pro Val Glu Pro Asp
Leu 85 90 95 Ala Val Ala Gly Ile His Ser Leu Pro Ser Thr Ser Ile
Ala Pro His 100 105 110 Ser Pro Ser Ser Pro Val Ala Ser Val Leu Leu
Gln Asp Thr Lys Pro 115 120 125 Thr Phe Glu Met Gln Gln Pro Ser Pro
Pro Ile Pro Pro Val His Pro 130 135 140 Asp Val Gln Leu Lys Thr Leu
Pro Phe Tyr Asp Val Leu Asp Val Leu 145 150 155 160 Ile Lys Pro Thr
Ser Leu Val Gln Ser Ser Ile Gln Arg Phe Gln Glu 165 170 175 Lys Phe
Phe Ile Phe Ala Leu Thr Pro Gln Gln Val Arg Glu Ile Cys 180 185 190
Ile Ser Arg Asp Phe Leu Pro Gly Gly Arg Arg Asp Tyr Thr Val Gln 195
200 205 Val Gln Leu Arg Leu Cys Leu Ala Glu Thr Ser Cys Pro Gln Glu
Asp 210 215 220 Asn Tyr Pro Asn Ser Leu Cys Ile Lys Val Asn Gly Lys
Leu Phe Pro 225 230 235 240 Leu Pro Gly Tyr Ala Pro Pro Pro Lys Asn
Gly Ile Glu Gln Lys Arg 245 250 255 Pro Gly Arg Pro Leu Asn Ile Thr
Ser Leu Val Arg Leu Ser Ser Ala 260 265 270 Val Pro Asn Gln Ile Ser
Ile Ser Trp Ala Ser Glu Ile Gly Lys Asn 275 280 285 Tyr Ser Met Ser
Val Tyr Leu Val Arg Gln Leu Thr Ser Ala Met Leu 290 295 300 Leu Gln
Arg Leu Lys Met Lys Gly Ile Arg Asn Pro Asp His Ser Lys 305 310 315
320 Ala Leu Ile Lys Glu Lys Leu Thr Ala Asp Pro Asp Ser Glu Ile Ala
325 330 335 Thr Thr Ser Leu Arg Val Ser Leu Met Cys Pro Leu Gly Lys
Met Arg 340 345 350 Leu Thr Ile Pro Cys Arg Ala Val Thr Cys Thr His
Leu Gln Cys Phe 355 360 365 Asp Ala Ala Leu Tyr Leu Gln Met Asn Glu
Lys Lys Pro Thr Trp Ile 370 375 380 Cys Pro Val Cys Asp Lys Lys Ala
Ala Tyr Glu Ser Leu Ile Leu Asp 385 390 395 400 Gly Leu Phe Met Glu
Ile Leu Asn Asp Cys Ser Asp Val Asp Glu Ile 405 410 415 Lys Phe Gln
Glu Asp Gly Ser Trp Cys Pro Met Arg Pro Lys Lys Glu 420 425 430 Ala
Met Lys Val Thr Ser Gln Pro Cys Thr Lys Val Glu Ser Ser Ser 435 440
445 Val Phe Ser Lys Pro Cys Ser Val Thr Val Ala Ser Asp Ala Ser Lys
450 455 460 Lys Lys Ile Asp Val Ile Asp Leu Thr Ile Glu Ser Ser Ser
Asp Glu 465 470 475 480 Glu Glu Asp Pro Pro Ala Lys Arg Lys Cys Ile
Phe Met Ser Glu Thr 485 490 495 Gln Ser Ser Pro Thr Lys Gly Val Leu
Met Tyr Gln Pro Ser Ser Val 500 505 510 Arg Val Pro Ser Val Thr Ser
Val Asp Pro Ala Ala Ile Pro Pro Ser 515 520 525 Leu Thr Asp Tyr Ser
Val Pro Phe His His Thr Pro Val Ser Ser Met 530 535 540 Ser Ser Asp
Leu Pro Gly Leu Asp Phe Leu Ser Leu Ile Pro Val Asp 545 550 555 560
Pro Gln Ser His Leu Thr Leu Asn Ser Lys Gln Tyr Val Cys His His 565
570 575 His Gln Pro Pro 580 37 1251 DNA Homo sapien 37 atgagcacca
agcagatcac ttgcaggtat tttatgcatg gtgtgtgtcg ggaaggaagt 60
cagtgcctat tctcacatga cttggcaaac agcaaaccgt ccaccatctg caagtactac
120 cagaagggct actgtgccta tggaactcgg tgcagatatg accacacgag
gccctctgct 180 gcagctggag gtgctgtggg caccatggcc cacagtgtgc
cctccccagc tttccacagt 240 cctcaccctc cttccgaggt cactgcatcc
attgtgaaaa ctaactcaca tgaacccgga 300 aagcgtgaaa agagaacatt
ggttcttaga gaccgaaatc tctctggcat ggctgaaagg 360 aagacccagc
cgagcatggt gagtaatcca ggcagctgca gcgaccccca gcccagcccc 420
gagatgaagc cgcattccta cctggatgcc atcaggagtg gccttgatga cgtggaggcc
480 agcagctcct acagcaacga gcagcagctg tgcccctacg cagctgctgg
ggagtgccgg 540 tttggggatg cctgtgtcta cctgcacggg gaggtgtgtg
aaatctgtag gctgcaagtc 600 ttgcacccat tcgacccaga gcagaggaag
gctcatgaaa agatctgcat gttgacgttc 660 gaacacgaga tggaaaaggc
ctttgccttc caggcaagcc aggacaaagt gtgcagtatc 720 tgcatggaag
tgatcctgga gaaggcctct gcttctgaga ggagatttgg gattctctcc 780
aattgcaatc acacgtactg tttgtcctgc atccggcagt ggcggtgtgc cgaacagttt
840 gaaaacccaa tcattaagtc ttgtccagaa tgccgtgtga tatcagagtt
tgtaattcca 900 agtgtgtatt gggtggaaga tcagaataaa aagaacgagt
tgattgaagc tttcaaacag 960 gggatgggga aaaaagcctg taaatacttt
gagcaaggca aggggacctg cccatttgga 1020 agcaaatgtc tttatcgcca
tgcttacccc gatgggcggc tagcagagcc tgagaaacct 1080 cggaaacagc
tcagttctca aggcactgtg aggttcttta attcagtgcg gctctgggat 1140
ttcatcgaga accgagaaag ccggcatgtc cccaacaatg aagatgtcga catgacagag
1200 ctcggggacc tcttcatgca cctttctgga gtggaatcat cagaacccta a 1251
38 416 PRT Homo sapien 38 Met Ser Thr Lys Gln Ile Thr Cys Arg Tyr
Phe Met His Gly Val Cys 1 5 10 15 Arg Glu Gly Ser Gln Cys Leu Phe
Ser His Asp Leu Ala Asn Ser Lys 20 25 30 Pro Ser Thr Ile Cys Lys
Tyr Tyr Gln Lys Gly Tyr Cys Ala Tyr Gly 35 40 45 Thr Arg Cys Arg
Tyr Asp His Thr Arg Pro Ser Ala Ala Ala Gly Gly 50 55 60 Ala Val
Gly Thr Met Ala His Ser Val Pro Ser Pro Ala Phe His Ser 65 70 75 80
Pro His Pro Pro Ser Glu Val Thr Ala Ser Ile Val Lys Thr Asn Ser 85
90 95 His Glu Pro Gly Lys Arg Glu Lys Arg Thr Leu Val Leu Arg Asp
Arg 100 105 110 Asn Leu Ser Gly Met Ala Glu Arg Lys Thr Gln Pro Ser
Met Val Ser 115 120 125 Asn Pro Gly Ser Cys Ser Asp Pro Gln Pro Ser
Pro Glu Met Lys Pro 130 135 140 His Ser Tyr Leu Asp Ala Ile Arg Ser
Gly Leu Asp Asp Val Glu Ala 145 150 155 160 Ser Ser Ser Tyr Ser Asn
Glu Gln Gln Leu Cys Pro Tyr Ala Ala Ala 165 170 175 Gly Glu Cys Arg
Phe Gly Asp Ala Cys Val Tyr Leu His Gly Glu Val 180 185 190 Cys Glu
Ile Cys Arg Leu Gln Val Leu His Pro Phe Asp Pro Glu Gln 195 200 205
Arg Lys Ala His Glu Lys Ile Cys Met Leu Thr Phe Glu His Glu Met 210
215 220 Glu Lys Ala Phe Ala Phe Gln Ala Ser Gln Asp Lys Val Cys Ser
Ile 225 230 235 240 Cys Met Glu Val Ile Leu Glu Lys Ala Ser Ala Ser
Glu Arg Arg Phe 245 250 255 Gly Ile Leu Ser Asn Cys Asn His Thr Tyr
Cys Leu Ser Cys Ile Arg 260 265 270 Gln Trp Arg Cys Ala Glu Gln Phe
Glu Asn Pro Ile Ile Lys Ser Cys 275 280 285 Pro Glu Cys Arg Val Ile
Ser Glu Phe Val Ile Pro Ser Val Tyr Trp 290 295 300 Val Glu Asp Gln
Asn Lys Lys Asn Glu Leu Ile Glu Ala Phe Lys Gln 305 310 315 320 Gly
Met Gly Lys Lys Ala Cys Lys Tyr Phe Glu Gln Gly Lys Gly Thr 325 330
335 Cys Pro Phe Gly Ser Lys Cys Leu Tyr Arg His Ala Tyr Pro Asp Gly
340 345 350 Arg Leu Ala Glu Pro Glu Lys Pro Arg Lys Gln Leu Ser Ser
Gln Gly 355 360 365 Thr Val Arg Phe Phe Asn Ser Val Arg Leu Trp Asp
Phe Ile Glu Asn 370 375 380 Arg Glu Ser Arg His Val Pro Asn Asn Glu
Asp Val Asp Met Thr Glu 385 390 395 400 Leu Gly Asp Leu Phe Met His
Leu Ser Gly Val Glu Ser Ser Glu Pro 405 410 415 39 738 DNA Homo
sapien 39 atgaaacgga ggaagcaaga tgaagggcag agggaaggct cctgcatggc
tgaggatgat 60 gctgtggaca tcgagcatga gaacaacaac cgctttgagg
agtatgagtg gtgtggacag 120 aagcggatac gggccaccac tctcctggaa
ggtggcttcc gaggctctgg cttcatcatg 180 tgcagcggca aagagaaccc
ggacagtgat gctgacttgg atgtggatgg ggatgacact 240 ctggagtatg
ggaagccaca atacacagag gctgatgtca tcccctgcac aggcgaggag 300
cctggtgaag ccaaggagag agaggcactt cggggcgcag tcctaaatgg cggccctccc
360 agcacgcgca tcacacctga gttctctaaa tgggccagtg atgagatgcc
atccaccagc 420 aatggtgaaa gcagcaagca ggaggccatg cagaagacct
gcaagaacag cgacatcgag 480 aaaatcaccg aagattcagc tgtgaccacg
tttgaggctc tgaaggctcg ggtcagagaa 540 cttgaacggc agctatctcg
tggggaccgt tacaaatgcc tcatctgcat ggactcgtac 600 tcgatgcccc
taacgtccat ccagtgttgg cacgtgcact gcgaggagtg ctggctgcgg 660
accctgggtg ccaagaagct ctgccctcag tgcaacacga tcacagcgcc cggagacctg
720 cggaggatct acttgtga 738 40 245 PRT Homo sapien 40 Met Lys Arg
Arg Lys Gln Asp Glu Gly Gln Arg Glu Gly Ser Cys Met 1 5 10 15 Ala
Glu Asp Asp Ala Val Asp Ile Glu His Glu Asn Asn Asn Arg Phe 20 25
30 Glu Glu Tyr Glu Trp Cys Gly Gln Lys Arg Ile Arg Ala Thr Thr Leu
35 40 45 Leu Glu Gly Gly Phe Arg Gly Ser Gly Phe Ile Met Cys Ser
Gly Lys 50 55 60 Glu Asn Pro Asp Ser Asp Ala Asp Leu Asp Val Asp
Gly Asp Asp Thr 65 70 75 80 Leu Glu Tyr Gly Lys Pro Gln Tyr Thr Glu
Ala Asp Val Ile Pro Cys 85 90 95 Thr Gly Glu Glu Pro Gly Glu Ala
Lys Glu Arg Glu Ala Leu Arg Gly 100 105 110 Ala Val Leu Asn Gly Gly
Pro Pro Ser Thr Arg Ile Thr Pro Glu Phe 115 120 125 Ser Lys Trp Ala
Ser Asp Glu Met Pro Ser Thr Ser Asn Gly Glu Ser 130 135 140 Ser Lys
Gln Glu Ala Met Gln Lys Thr Cys Lys Asn Ser Asp Ile Glu 145 150 155
160 Lys Ile Thr Glu Asp Ser Ala Val Thr Thr Phe Glu Ala Leu Lys Ala
165 170 175 Arg Val Arg Glu Leu Glu Arg Gln Leu Ser Arg Gly
Asp Arg Tyr Lys 180 185 190 Cys Leu Ile Cys Met Asp Ser Tyr Ser Met
Pro Leu Thr Ser Ile Gln 195 200 205 Cys Trp His Val His Cys Glu Glu
Cys Trp Leu Arg Thr Leu Gly Ala 210 215 220 Lys Lys Leu Cys Pro Gln
Cys Asn Thr Ile Thr Ala Pro Gly Asp Leu 225 230 235 240 Arg Arg Ile
Tyr Leu 245 41 1425 DNA Homo sapien 41 atgtcgtcag aagatcgaga
agctcaggag gatgaattgc tggccctggc aagtatttac 60 gatggagatg
aatttagaaa agcagagtct gtccaaggtg gagaaaccag gatctatttg 120
gatttgccac agaatttcaa gatatttgtg agcggcaatt caaatgagtg tctccagaat
180 agtggctttg aatacaccat ttgctttctg cctccacttg tgctgaactt
tgaactgcca 240 ccagattatc catcctcttc cccaccttca ttcacactta
gtggcaaatg gctgtcacca 300 actcagctat ctgctctatg caagcactta
gacaacctat gggaagaaca ccgtggcagc 360 gtggtcctgt ttgcctggat
gcaatttctt aaggaagaga ccctagcata cttgaatatt 420 gtctctcctt
ttgagctcaa gattggttct cagaaaaaag tgcagagaag gacagctcaa 480
gcttctccca acacagagct agattttgga ggagctgctg gatctgatgt agaccaagag
540 gaaattgtgg atgagagagc agtgcaggat gtggaatcac tgtcaaatct
gatccaggaa 600 atcttggact ttgatcaagc tcagcagata aaatgcttta
atagtaaatt gttcctgtgc 660 agtatctgtt tctgtgagaa gctgggtagt
gaatgcatgt acttcttgga gtgcaggcat 720 gtgtactgca aagcctgtct
gaaggactac tttgaaatcc agatcagaga tggccaggtt 780 caatgcctca
actgcccaga accaaagtgc ccttcggtgg ccactcctgg tcaggtcaaa 840
gagttagtgg aagcagagtt atttgcccgt tatgaccgcc ttctcctcca gtcctccttg
900 gacctgatgg cagatgtggt gtactgcccc cggccgtgct gccagctgcc
tgtgatgcag 960 gaacctggct gcaccatggg tatctgctcc agctgcaatt
ttgccttctg tactttgtgc 1020 aggttgacct accatggggt ctccccatgt
aaggtgactg cagagaaatt aatggactta 1080 cgaaatgaat acctgcaagc
ggatgaggct aataaaagac ttttggatca aaggtatggt 1140 aagagagtga
ttcagaaggc actggaagag atggaaagta aggagtggct agagaagaac 1200
tcaaagagct gcccatgttg tggaactccc atagagaaat tagacggatg taacaagatg
1260 acatgtactg gctgtatgca atatttctgt tggatttgca tgggttctct
ctctagagca 1320 aacccttaca aacatttcaa tgaccctggt tcaccatgtt
ttaaccggct gttttatgct 1380 gtggatgttg acgacgatat ttgggaagat
gaggtagaag actag 1425 42 474 PRT Homo sapien 42 Met Ser Ser Glu Asp
Arg Glu Ala Gln Glu Asp Glu Leu Leu Ala Leu 1 5 10 15 Ala Ser Ile
Tyr Asp Gly Asp Glu Phe Arg Lys Ala Glu Ser Val Gln 20 25 30 Gly
Gly Glu Thr Arg Ile Tyr Leu Asp Leu Pro Gln Asn Phe Lys Ile 35 40
45 Phe Val Ser Gly Asn Ser Asn Glu Cys Leu Gln Asn Ser Gly Phe Glu
50 55 60 Tyr Thr Ile Cys Phe Leu Pro Pro Leu Val Leu Asn Phe Glu
Leu Pro 65 70 75 80 Pro Asp Tyr Pro Ser Ser Ser Pro Pro Ser Phe Thr
Leu Ser Gly Lys 85 90 95 Trp Leu Ser Pro Thr Gln Leu Ser Ala Leu
Cys Lys His Leu Asp Asn 100 105 110 Leu Trp Glu Glu His Arg Gly Ser
Val Val Leu Phe Ala Trp Met Gln 115 120 125 Phe Leu Lys Glu Glu Thr
Leu Ala Tyr Leu Asn Ile Val Ser Pro Phe 130 135 140 Glu Leu Lys Ile
Gly Ser Gln Lys Lys Val Gln Arg Arg Thr Ala Gln 145 150 155 160 Ala
Ser Pro Asn Thr Glu Leu Asp Phe Gly Gly Ala Ala Gly Ser Asp 165 170
175 Val Asp Gln Glu Glu Ile Val Asp Glu Arg Ala Val Gln Asp Val Glu
180 185 190 Ser Leu Ser Asn Leu Ile Gln Glu Ile Leu Asp Phe Asp Gln
Ala Gln 195 200 205 Gln Ile Lys Cys Phe Asn Ser Lys Leu Phe Leu Cys
Ser Ile Cys Phe 210 215 220 Cys Glu Lys Leu Gly Ser Glu Cys Met Tyr
Phe Leu Glu Cys Arg His 225 230 235 240 Val Tyr Cys Lys Ala Cys Leu
Lys Asp Tyr Phe Glu Ile Gln Ile Arg 245 250 255 Asp Gly Gln Val Gln
Cys Leu Asn Cys Pro Glu Pro Lys Cys Pro Ser 260 265 270 Val Ala Thr
Pro Gly Gln Val Lys Glu Leu Val Glu Ala Glu Leu Phe 275 280 285 Ala
Arg Tyr Asp Arg Leu Leu Leu Gln Ser Ser Leu Asp Leu Met Ala 290 295
300 Asp Val Val Tyr Cys Pro Arg Pro Cys Cys Gln Leu Pro Val Met Gln
305 310 315 320 Glu Pro Gly Cys Thr Met Gly Ile Cys Ser Ser Cys Asn
Phe Ala Phe 325 330 335 Cys Thr Leu Cys Arg Leu Thr Tyr His Gly Val
Ser Pro Cys Lys Val 340 345 350 Thr Ala Glu Lys Leu Met Asp Leu Arg
Asn Glu Tyr Leu Gln Ala Asp 355 360 365 Glu Ala Asn Lys Arg Leu Leu
Asp Gln Arg Tyr Gly Lys Arg Val Ile 370 375 380 Gln Lys Ala Leu Glu
Glu Met Glu Ser Lys Glu Trp Leu Glu Lys Asn 385 390 395 400 Ser Lys
Ser Cys Pro Cys Cys Gly Thr Pro Ile Glu Lys Leu Asp Gly 405 410 415
Cys Asn Lys Met Thr Cys Thr Gly Cys Met Gln Tyr Phe Cys Trp Ile 420
425 430 Cys Met Gly Ser Leu Ser Arg Ala Asn Pro Tyr Lys His Phe Asn
Asp 435 440 445 Pro Gly Ser Pro Cys Phe Asn Arg Leu Phe Tyr Ala Val
Asp Val Asp 450 455 460 Asp Asp Ile Trp Glu Asp Glu Val Glu Asp 465
470 43 6 PRT Artificial Sequence nuclear localization signal 43 His
Lys Ala Val Lys Arg 1 5 44 7 PRT Artificial Sequence nuclear
localization signal 44 Arg Leu Lys Ile Thr Lys Lys 1 5 45 6 PRT
Artificial Sequence putative protein kinase phosphorylation site 45
Arg Pro Arg Ser Phe Thr 1 5 46 7 PRT Artificial Sequence protein
kinase phosphorylation site consensus sequence 46 Arg Xaa Arg Xaa
Xaa Ser Thr 1 5 47 24 PRT Artificial Sequence consensus sequence 47
Ser Thr Xaa Pro Xaa Ser Pro Xaa Ser Xaa Pro Xaa Ser Pro Xaa Xaa 1 5
10 15 Xaa Gly Xaa Xaa Xaa Xaa Ser Asp 20 48 9 PRT Artificial
Sequence neuralized homology repeat domain 48 Leu Pro Lys Tyr Ala
Cys Pro Asp Leu 1 5
* * * * *