U.S. patent application number 10/300834 was filed with the patent office on 2003-04-24 for novel semaphorin gene: semaphorin y.
This patent application is currently assigned to Sumitomo Pharmaceuticals Company, Limited. Invention is credited to Kikuchi, Kaoru, Kimura, Toru.
Application Number | 20030077669 10/300834 |
Document ID | / |
Family ID | 26527563 |
Filed Date | 2003-04-24 |
United States Patent
Application |
20030077669 |
Kind Code |
A1 |
Kimura, Toru ; et
al. |
April 24, 2003 |
Novel Semaphorin gene: Semaphorin Y
Abstract
The present invention provides Semaphorin Y inhibiting neurite
outgrowth, and a gene therefor, as well as other Semaphorins
hybridizing to said Semaphorin Y gene, modified proteins or partial
peptides of said Semaphorin Y, antibodies against said Semaphorin
Y, antisense nucleotides against said Semaphorin Y gene, and the
use of such substances as pharmaceutical or diagnostic agents or
laboratory reagents. The present invention further provides a
method of screening for Semaphorin Y antagonists employing said
Semaphorin Y, Semaphorin Y antagonists obtained by said screening
method, pharmaceutical agents comprising such antagonists, and
transgenic animals involving said Semaphorin Y.
Inventors: |
Kimura, Toru; (Kusatsu-shi,
JP) ; Kikuchi, Kaoru; (Takarazuka-shi, JP) |
Correspondence
Address: |
BIRCH STEWART KOLASCH & BIRCH
PO BOX 747
FALLS CHURCH
VA
22040-0747
US
|
Assignee: |
Sumitomo Pharmaceuticals Company,
Limited
|
Family ID: |
26527563 |
Appl. No.: |
10/300834 |
Filed: |
November 21, 2002 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10300834 |
Nov 21, 2002 |
|
|
|
09254594 |
May 11, 1999 |
|
|
|
09254594 |
May 11, 1999 |
|
|
|
PCT/JP97/03167 |
Sep 9, 1997 |
|
|
|
Current U.S.
Class: |
435/7.2 ;
435/320.1; 435/325; 435/69.1; 530/350; 536/23.5 |
Current CPC
Class: |
A61K 38/00 20130101;
C07K 14/4703 20130101; A01K 2217/05 20130101 |
Class at
Publication: |
435/7.2 ;
435/69.1; 435/320.1; 435/325; 530/350; 536/23.5 |
International
Class: |
G01N 033/53; G01N
033/567; C07H 021/04; C12P 021/02; C12N 005/06; C07K 014/47 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 11, 1996 |
JP |
263565/1996 |
Aug 8, 1997 |
JP |
227220/1997 |
Claims
1. An isolated polypeptide comprising the amino acid sequence shown
in SEQ ID No: 3.
2. An isolated polypeptide comprising the amino acid sequence shown
in SEQ ID NO: 6.
3. A polypeptide obtained by expressing in a cell a nucleic acid
comprising a polynucleotide having a nucleotide sequence selected
from the group consisting of: (a) the nucleotide sequence of SEQ ID
NO: 1, (b) the nucleotide sequence of SEQ ID NO: 2, (c) the
nucleotide sequence of SEQ ID NO: 4, (d) the nucleotide sequence of
SEQ ID NO: 5, (e) a cDNA sequence encoding the amino acid sequence
of SEQ ID NO:3, and (f) a cDNA sequence encoding the amino acid
sequence of SEQ ID NO: 6.
4. A polypeptide obtained by expressing in a cell a nucleic acid
comprising a cDNA that i) specifically hybridizes to the antisense
strand of the nucleotide sequence of SEQ ID NO: 1, SEQ ID NO: 2,
SEQ ID NO: 4 or SEQ ID NO: 5 under hybridization conditions of a
buffer comprising 45%(v/v) formamide, 5.times.SSPE at 42.degree.
C., and washing after hybridization with a buffer comprising
2.times.SSPE at 55.degree. C.; and ii) encodes a protein having
biological activity of inhibiting neurite outgrowth of neurons of
the peripheral nervous system or of collapsing growth cones of
neurons of the peripheral nervous system or both.
5. The polypeptide according to claim 4, wherein said nucleic acid
originates from a mammal.
6. An isolated polypeptide comprising six or more contiguous amino
acids of SEQ ID NO: 3.
7. An isolated polypeptide comprising six or more contiguous amino
acids of SEQ ID NO: 6.
8. A method for screening for an antagonist of Semaphorin Y,
comprising the steps of: adding a polypeptide comprising the amino
acid sequence of SEQ ID NO: 3 or SEQ ID NO: 6 to an assay system
for Semaphorin Y activity, and adding a test substance to said
assay system, and assaying for Semaphorin Y activity of collapsing
growth cones of neurons, wherein inhibition of the Semaphorin Y
activity which results from the addition of the test substance
indicates that said test substance is an antagonist of Semaphorin
Y.
9. A method for screening for an antagonist of Semaphorin Y,
comprising the steps of: adding a polypeptide according to claim 3
or claim 4 to an assay system for Semaphorin Y activity, and adding
a test substance to said assay system, and assaying for Semaphorin
Y activity of collapsing growth cones of neurons, wherein
inhibition of the Semaphorin Y activity which results from the
addition of the test substance indicates that said test substance
is an antagonist of Semaphorin Y.
Description
TECHNICAL FIELD
[0001] The present invention relates to Semaphorin Y, a novel
Semaphorin belonging to the Semaphorin family, and use of
Semaphorin Y for pharmaceutical or diagnostic agents or laboratory
reagents. More particularly, it relates to Semaphorin Y inhibiting
neurite outgrowth, and a gene therefor, as well as other
Semaphorins hybridizing to said Semaphorin Y gene, modified
proteins or partial peptides of said Semaphorin Y, antibodies
against said Semaphorin Y, antisense nucleotides against said
Semaphorin Y gene, antagonists of said Semaphorin Y, transgenic
animals, and their use as pharmaceutical or diagnostic agents or
laboratory reagents.
BACKGROUND ART
[0002] It is widely known that a central nervous system
(CNS)-neuron in higher organisms such as human is not capable of
regeneration once injured. Therefore, one who has received an
injury on his (her) spinal cord due to, for example, a traffic
accident, is compelled to spend the rest of his (her) life in a
hemiplegic state. On the contrary, it is known that a peripheral
nervous system (PNS)-neuron retains a vigorous regeneration ability
even in those higher organisms, and therefore, neurons in a limb,
when disconnected, can gradually regenerate with a concomitant
recovery of their function.
[0003] In the early nineteen-eighties, a group of Aguayo et al.
found that when PNS-neuron is experimentally grafted into an
injured CNS-neuron in a higher organism, axon growth of CNS-neuron
is induced. This observation demonstrates that CNS-neuron in higher
organisms which had been generally considered not to have a
regeneration ability can regenerate if a suitable environment is
provided (Nature, 284, 264-265 (1980), Science, 214, 931-933
(1981)). That report suggests a possibility that in CNS of higher
organisms, there may exist a factor, namable "CNS-neuron
regeneration inhibitor", which inhibits the regeneration of
CNS-neuron, and that a release from such inhibition may allow the
regeneration of CNS-neurons. This suggestion paved the way for a
CNS-neuron regeneration therapy.
[0004] In 1988, a group of Schwab et al. demonstrated that there
exited such CNS-neuron regeneration inhibitor among proteins
derived from CNS myelin. They also succeeded in purifying, though
partially, a protein having said CNS-neuron regeneration inhibition
activity) and named this protein fraction NI35/250 (Annu. Rev.
Neurosci., 16, 565-595 (1993)), although no one has succeeded in
its isolation, identification and gene cloning yet. In addition,
they immunized animals with the partial purified NI35/250, and
succeeded in obtaining an antibody (IN-1) having a neutralizing
activity. This antibody is capable of recognizing a band for
NI35/250 in Western blotting, and capable of staining, in an
immunostaining, the region to which NI35/250 is supposed to be
distributed. Furthermore, they demonstrated that administration of
this antibody to an animal experimentally received an injury on its
spinal cord has promoted regeneration of axons in spinal cord,
though partially, within 2-3 weeks, and restored its function
within 2-3 months (Nature, 343, 269-272 (1990), Nature, 378,
498-501 (1995)). These findings are of great value, because they
experimentally demonstrated that there existed a CNS-neuron
regeneration inhibitor as suggested by Aguayo et al. (supra) and
that CNS-neuron can be regenerated by inhibiting the activity of
said inhibitor. The above antibody is, however, directed not to
human but to rat NI35/250, and exhibits a low stability and
specificity. In addition, although regeneration of CNS-neuron was
observed as described above by administering said antibody, its
effect was so partial and incomplete that not all of the motor
functions could be restored. It is, therefore, believed essential
in solving these problems to identify the gene for NI35/250 or
corresponding CNS-neuron regeneration inhibitor, and, based on
knowledges of molecular biology, neuroscience and the like, develop
an antagonist more effectively inhibiting the CNS-neuron
regeneration inhibition activity, or develop a method for
inhibiting the expression of the gene for said regeneration
inhibitor.
[0005] Apart from the above, the nervous system, whether it is
central or peripheral, requires formation of a complicated neural
network among neurons or between neurons and peripheral receivers
or effectors during development, that is, in the stage of embryo or
fetus, in order to precisely carry out its principal functions,
i.e., to transfer and process the information. To establish the
neural network, an ingenious mechanism is necessary, which
precisely guides a growing neurite to the target site locating
remote therefrom.
[0006] It has been hitherto believed that a factor which positively
controls the neurite outgrowth, such as neurite growth promoter and
neurite growth attractant may play a major role in the formation of
the neural network. However, it is now being demonstrated by recent
studies on the mechanism of the network formation that the opposite
factor, that is, a negative factor having an outgrowth inhibition
activity is important for an accurate guidance (Cell, 78, 353-356
(1994)).
[0007] A representative factor having such an outgrowth inhibition
activity is a protein called "Semaphorin". Semaphorin firstly
discovered is Fasciclin IV found in grasshopper. Collapsin
(latterly named Collapsin I) was subsequently discovered in chick
(Cell, 75, 217-227 (1993); Neuron, 9, 831-845 (1992)). To date,
more than 10 genes belonging to the Semaphorin family have been
reported in a wide range of species covering insects such as
drosophila and beetle, human, and viruses (Cell, 81, 471-474
(1995)). These Semaphorins characteristically contain in their
amino acid sequences similar structures called semaphorin domains
each consisting of about 500 amino acids (Neuron, 14, 941-948
(1995); Cell, 75, 1389-1399 (1993)). However, the homologies of the
primary amino acid sequences in semaphorin domains among these
Semaphorin genes are 80-20%, and not necessarily high.
[0008] Of these Semaphorins, functions have been verified for only
a few, including, for example, Fasciclin IV of grasshopper,
Semaphorins I and II of drosophila, Collapsin of chick, and
Semaphorin III which corresponds to Collapsin in mammals. All of
these Semaphorins are known to inhibit neurite outgrowth or
synapsis formation. In particular, Semaphorin III has been reported
to have an activity collapsing in a short time the growth cone of
cultured neuron (growth-cone collapse activity) in vitro (Neuron,
14, 941-948 (1995); Neuron, 14, 949-959 (1995); Cell, 75, 631-639
(1995); Cell, 75, 1389-1399 (1993); Cell, 75, 217-227 (1993);
Neuron, 9, 831-845 (1992)).
[0009] Although it is now being demonstrated, as described above,
that known Semaphorins have a growth-cone collapse activity and a
neurite outgrowth inhibition activity during development, and play
a role in giving an accurate guidance to neuron, it is not evident
at present whether or not their Semaphorins exert some function not
only during development but also in the adult, and less evident
whether or not Semaphorins play a role as a CNS-neuron regeneration
inhibitor. Of course, since known Semaphorins have been shown to be
a negative guidance factor inhibiting neurite outgrowth, it would
not be unreasonable to consider said Semaphorins as a candidate for
a CNS-neuron regeneration inhibitor (Nature, 378, 439-440 (1995)).
However, it has been shown by in vitro experiments that Semaphorin
III (Sema III), only one Semaphorin of higher organisms of which
function has been analyzed, exerts its neurite-outgrowth inhibition
activity on a sensory neuron and sympathetic neuron both of which
are peripheral, but not on a retinal neuron which is central (Cell,
75, 217-227 (1993)). In addition, Northern analysis on the
distribution of Sema III expression in the adult conducted by the
present inventors has revealed that it is expressed mainly in
peripheral tissues (see Reference example 2 below). It is therefore
hardly believed that Sema III having such features has a function
as a CNS-neuron regeneration inhibitor.
PROBLEM TO BE SOLVED BY THE INVENTION
[0010] The present invention aims to provide Semaphorin Y, a novel
Semaphorin belonging to the Semaphorin family, and a gene therefor,
and to provide pharmaceutical agents for neural diseases, in
particular for regeneration of CNS-neuron, and related diagnostic
agents or laboratory reagents. More specifically, the present
invention aims to provide Semaphorin Y inhibiting neurite outgrowth
and a gene therefor, as well as other Semaphorins hybridizing to
said Semaphorin Y gene, modified proteins or partial peptides of
said Semaphorin Y, antibodies against said Semaphorin Y, antisense
nucleotides against said Semaphorin Y gene, and use of such
substances as pharmaceutical or diagnostic agents or laboratory
reagents. The present invention further aims to provide a method of
screening for Semaphorin Y antagonists empolying said Semaphorin Y,
Semaphorin Y antagonists obtained by said screening method,
pharmaceutical agents comprising such antagonists, and transgenic
animals involving Semaphorin Y.
MEANS OF SOLVING THE PROBLEM
[0011] In order to provide pharmaceutical agents for neural
diseases, in particular for regeneration of CNS-neuron, and related
diagnostic agents or laboratory reagents, the present inventors
have planed to identify a novel Semaphorin which has not yet been
cloned. In particular, the present inventors have paid their
attention to the similarity between the in vitro activities of the
above-described NI35/250 and Semaphorin, i.e., to the fact that
NI35/250 has a growth-cone collapse activity and a neurite-growth
inhibition activity in vitro (J. Neurosci., 8, 2381-2393 (1988);
Science, 259, 80 (1993)), while known Semaphorins similarly possess
a neurite-growth inhibition activity, and particularly Semaphorin
III has also a growth-cone collapse activity. This suggested to the
inventors the possibility that unknown Semaphorins which have not
yet been identified may include the one inhibiting regeneration of
CNS-neuron. Specifically, the present inventors' idea was that
Semaphorin, which is characterized in that 1) it is widely
expressed throughout the CNS of adult where regeneration of neuron
(or neurite outgrowth) is inhibited, but 2) it is poorly expressed
in other tissues such as peripheral tissues in the adult, has not
been identified yet, and if one can identify a new unknown
Semaphorin having such characteristics, the Semaphorin might be
involved in inhibition of regeneration of CNS-neuron.
[0012] First of all, the inventors have closely searched DNA
database on the basis of the amino acids sequence relatively well
conserved among previously reported Semaphorin genes. Specifically,
a DNA sequence has been searched through EST (Expressed Sequence
Tags) database, which is a gene not expressed in peripheral tissues
but expressed in the postnatal brain and which encodes an amino
acid sequence relatively well conserved among Semaphorins. As a
consequence, a DNA fragment R59527 was identified, which encodes,
as a partial sequence, a sequence consisting of seven amino acids:
Gln (or Arg)-Asp-Pro-Tyr-Cys-Ala (or Gly)-Trp. The R59527 gave a
sequence information as to only 238 bases, and furthermore only
several percent thereof could be translated into an amino acid
sequence common to those of known Semaphorins. In addition, the
reading frame could not be determined because of the presence of
sequence not definitely determined in R59527. It was, therefore,
utterly impossible at that stage to conclude that the base sequence
of R59527 is part of a novel Semaphorin. We have, however, finally
succeeded in cloning a novel Semaphorin gene by carrying out the
following procedures: synthesizing DNA primers on the basis of that
sequence information; conducting PCR with said primers using cDNAs
prepared from a human hippocampal cDNA library as templates to
obtain a novel DNA fragment (SEQ ID NO: 7) consisting of 170 bases;
labeling the DNA fragment with .sup.32P to synthesize a DNA probe;
and screening rat and human cDNA libraries with that probe. We
named this novel Semaphorin "Semaphorin Y".
[0013] Subsequent analysis revealed that Semaphorin Y is a novel
Semaphorin at which we aimed, since it was widely expressed in CNS
in the adult, whereas among peripheral tissues the expression could
be observed only in limited tissues.
[0014] Semaphorin Y of the present invention having such
characteristics appears to be involved in inhibition of
regeneration of CNS-neuron in the adult. Semaphorin Y may be used
to screen for Semaphorin Y antagonists, and antagonists identified
in such screening system are expected to promote regeneration of
CNS-neuron. Similarly, antisense DNAs or RNAs against Semaphorin Y
gene are also expected to promote regeneration of CNS-neuron as the
above antagonists do.
[0015] In addition, in view of the fact that Semaphorin Y of the
present invention inhibits neurite outgrowth, it may be used as a
therapeutic or diagnostic agent for pains or immune diseases such
as atopic dermatitis, by administering it to peripheral tissues,
which results in the inhibition of neurite outgrowth of PNS-neuron.
Furthermore, Semaphorin Y is a novel Semaphorin belonging to the
Semaphorin family of which expression distribution is
unconventionally characteristic as described above, and also has a
characteristic in that it does not contain any Ig domains commonly
found among hitherto reported Semaphorins of vertebrates.
Semaphorin Y may, therefore, serve as an important research
material or a laboratory reagent.
[0016] The present invention has been completed on the basis of the
above findings.
[0017] Thus, the gist of the present invention is as follows:
[0018] (1) a gene encoding the following protein (a) or (b):
[0019] (a) Semaphorin Y protein comprising the amino acid sequence
shown in SEQ ID NO: 3 or 6,
[0020] (b) a protein which comprises an amino acid sequence wherein
one or more amino acids are deleted, substituted and/or added in
the amino acid sequence shown in SEQ ID NO: 3 or 6, and which
protein inhibits neurite outgrowth;
[0021] (2) a gene comprising the following DNA (a) or (b):
[0022] (a) Semaphorin Y DNA comprising the base sequence shown in
SEQ ID NO: 1, 2, 4, or 5,
[0023] (b) DNA which hybridizes under stringent conditions to DNA
comprising the base sequence shown in SEQ ID NO: 1, 2, 4, or 5, and
which encodes a protein inhibiting neurite outgrowth;
[0024] (3) a gene comprising DNA which hybridizes under stringent
conditions to DNA comprising the base sequence shown in SEQ ID NO:
7, and which encodes a protein having a semaphorin domain;
[0025] (4) a protein obtained by expressing the gene of any one of
the above items (1) to (3);
[0026] (5) a gene comprising DNA which encodes a protein comprising
an amino acid sequence in which one or more amino acids are
deleted, substituted and/or added in the protein shown in SEQ ID
NO: 3 or 6, wherein said protein promotes neurite outgrowth;
[0027] (6) a protein obtained by expressing the gene of the above
item (5);
[0028] (7) DNA which is cloned from a human cDNA library or a human
genomic library, and which hybridizes under stringent conditions to
DNA comprising at least part of DNA consisting of the base sequence
shown in SEQ ID NO: 1 or 4;
[0029] (8) an expression plasmid which expresses either the gene of
any one of the above items (1) to (3) and (5), or DNA of the above
item (7);
[0030] (9) a transformant transformed with the expression plasmid
of the above item (8);
[0031] (10) a process for producing a recombinant protein, which
process comprises culturing the transformant of the above item (9),
and recovering the recombinant protein expressed;
[0032] (11) a peptide comprising at least six amino acids of the
protein of the above item (4) or (6);
[0033] (12) a peptide of the above item (11) which promotes neurite
outgrowth;
[0034] (13) a peptide of the above item (11) characterized in that
it contains aspartic acid residue at position 198 of the amino acid
sequence shown in SEQ ID NO: 6 or an amino acid residue
corresponding to the position of said aspartic acid residue;
[0035] (14) an antisense nucleotide, or chemically modified variant
thereof, which is directed against a segment comprising at least
eight or more bases in the gene of any one of the above items (1)
to (3), or in DNA of the above item (7);
[0036] (15) an antisense nucleotide or chemically modified variant
thereof of the above item (14), characterized in that it inhibits
expression of the protein of the above item (4);
[0037] (16) an antibody against the protein of the above item (4)
or (6), or against the peptide of any one of the above items (11)
to (13);
[0038] (17) a pharmaceutical agent comprising, as an active
ingredient, the gene of any one of the above items (1) to (3) and
(5), DNA of the above item (7), the protein of the above item (4)
or (6), the peptide of any one of the above items (11) to (13), the
antisense nucleotide or chemically modified variant thereof of the
above item (14) or (15), or the antibody of the above item
(16);
[0039] (18) a method of screening for Semaphorin Y antagonists,
characterized in that it employs the protein of the above item
(4);
[0040] (19) Semaphorin Y antagonist obtained by the screening
method of the above item (18);
[0041] (20) Semaphorin Y antagonist of the above item (19) which
comprises the protein of the above item (6), the peptide of any one
of the above items (11) to (13), or the antibody of the above item
(16);
[0042] (21) a CNS-neuron regeneration promoter, characterized in
that it contains at least one of the antisense nucleotides or
chemically modified variants thereof of the above item (14) or
(15), or Semaphorin Y antagonists of the above item (19) or
(20);
[0043] (22) a neurite outgrowth inhibitor for PNS-neuron,
characterized in that it contains at least one of the proteins of
the above item (4); and
[0044] (23) a transgenic animal in which either the gene of any one
of the above items (1) to (3) and (5), or DNA of the above item (7)
has been artificially inserted into its chromosome, or has been
knocked out.
MODE FOR CARRYING OUT THE INVENTION
[0045] The 1st embodiment of the present invention is a gene which
encodes Semaphorin Y comprising the amino acid sequence shown in
SEQ ID NO: 3 or 6, or a gene encoding a protein which comprises an
amino acid sequence wherein one or more amino acids are deleted,
substituted and/or added in the amino acid sequence of the above
Semaphorin Y, and which protein inhibits neurite outgrowth. The 2nd
embodiment of the present invention is Semaphorin Y gene comprising
the base sequence shown in SEQ ID NO: 1, 2, 4, or 5, or a gene
which hybridizes under stringent conditions to such Semaphorin Y
gene and which encodes a protein inhibiting neurite outgrowth.
These genes are explained below in order.
[0046] 1) Gene Encoding Semaphorin Y (Semaphorin Y Gene)
[0047] Of the above-mentioned genes, "a gene which encodes
Semaphorin Y protein comprising the amino acid sequence shown in
SEQ ID NO: 3" or "Semaphorin Y gene comprising the base sequence
shown in SEQ ID NO: 1 or 2" is a gene encoding the rat Semaphorin Y
of the present invention, while "a gene which encodes Semaphorin Y
protein comprising the amino acid sequence shown in SEQ ID NO: 6"
or "Semaphorin Y gene comprising the base sequence shown in SEQ ID
NO: 4 or 5" is a gene encoding the human Semaphorin Y of the
present invention. Among these genes, those shown in SEQ ID NOs: 2
and 5 correspond open reading frames for rat and human types of
Semaphorin Y, respectively. Such genes may be cloned, as described
in Example 1, by screening a cDNA library derived from CNS tissues
using a probe (for example, a DNA probe having the base sequence
shown in SEQ ID NO: 7) prepared on the basis of the sequence of
"R59527" found in EST database. Particular techniques for such
cloning may be found in the standard texts such as "Molecular
Cloning, 2nd ed.", Cold Spring Harbor Laboratory Press (1989). The
base sequence of the cloned DNA may also be determined by
conventional methods, for example, using a sequencing kit
commercially available.
[0048] Alternatively, after publication of the base sequence of rat
and human Semaphorin Y cDNAs of the present invention, one skilled
in the art can also easily clone the full-length genes encoding rat
and human types of Semaphorin Y by using part of said cDNA as a
probe, without using cloning methods as described above.
[0049] 2) Gene Encoding Modified Protein of Semaphorin Y
[0050] Of the above-mentioned genes, "a gene encoding a protein
which comprises an amino acid sequence wherein one or more amino
acids are deleted, substituted and/or added in the amino acid
sequence of the above Semaphorin Y, and which protein inhibits
neurite outgrowth" refers to a gene encoding a so-called "modified
proteins" of Semaphorin Y which inhibits neurite outgrowth. Those
skilled in the art may easily obtain a gene encoding such protein,
for example, by site-directed mutagenesis (Methods in Enzymology,
100, 448- (1983)) or PCR method (Molecular Cloning, 2nd ed.,
Chapter 15, Cold Harbor Laboratory Press (1989); "PCR A Practical
Approach", IRL Press, 200-210 (1991)). In this context, the number
of amino acid residues to be deleted, substituted and/or added is
to be such a number that permits the deletion, substitution and/or
addition by well-known methods such as site-directed mutagenesis
described above.
[0051] For the purpose of the present invention, the phrase
"inhibiting neurite outgrowth" means that the protein has the
collapse activity on growth cone of neuron, or that the protein has
the neurite-outgrowth inhibition activity. These activities may be
measured with a test substance such as an expression product of DNA
encoding Semaphorin Y or modified protein thereof, for example, in
the following manner:
[0052] Since Semaphorin Y is a membrane protein, it exists in the
cell membrane of the cells transformed with Semaphorin Y gene. The
activities of the above test substance may, therefore, easily be
measured by using, as a test material, the membrane fraction of the
transformed cells.
[0053] Examples of activity measurement include measurement of
collapse activity on growth cone of neuron (M. Igarashi et al.,
Science, vol. 259, pp. 77-79 (1993)), or measurement of
neurite-outgrowth inhibition activity (e.g., J. A. Davies et al.,
Neuron, vol. 2, pp. 11-20 (1990) and M. Bastmeyer, J. Neurosci.,
vol. 11, pp. 626-640 (1991)). A method of measuring the growth-cone
collapse activity is described in detail in literature (M. Igarashi
et al., Science, vol. 259, pp. 77-79 (1993)). Briefly, the
measurement may be carried out by a method in which cells
expressing a test substance such as Semaphorin Y is homogenized,
and the homogenate containing the cell membrane fraction or the
purified membrane fraction is used (E. C. Cox et al., Neuron, vol.
2, pp. 31-37 (1990)), or by a method in which a protein extracted
from the membrane fraction is reconstituted in a liposome and the
liposome is used as a test material (C. E. Bandtlow, Science, vol.
259, pp. 80-84 (1993)). In order to measure the growth-cone
collapse activity in practice using these materials, a test
substance such as Semaphorin Y, for example, in one of the forms as
describe above is added to neurons cultured under conventional
conditions (e.g., "Culturing, Nerve Cells" edited by Banker et al.,
MIT Press (1991)) in a container coated with a substance promoting
the neurite outgrowth and the growth-cone formation such as
laminin, collagen, polylysine or polyornithine. After the addition,
when a sufficient time has passed to occur collapse of growth cone
(typically from 30 minutes to one hour after the addition), those
neurons are fixed with 1% glutaraldehyde or the like, and the
number of the growth cones which have been collapsed is counted
under a microscope. In this measurement, it is important that
another sample is used as a control, which is prepared from cells
not expressing the test substance such as Semaphorin Y according to
the completely same procedures as those used for the test
substance-expressing cells. Typically, normalization of the samples
is conducted on the basis of the total amounts of protein included
within the samples. To measure the neurite-outgrowth inhibition
activity, part of the surface of a micropore filter or a culture
container made of glass or plastics is coated with a test substance
such as Semaphorin Y prepared as described above, and the activity
is indicated, for example, by the inability of neurons cultured
under conventional conditions to adhere to the coated area, or by a
remarkable decrease in the rate of neurite outgrowth on the coated
area, or by the inability of invasion of growing neurites from the
outside of the coated area into the coated area because of its
stopping on the border between the coated and non-coated areas or
its avoidance from the coated area. When a cluster of cells
expressing a test substance is co-cultured with neurons in a
collagen gel, the inability of outgrowing neurite to enter the
cluster of cells expressing the test substance may also be used as
an indicator (A. Sophia et al., Cell, vol. 81, 621-629 (1995)).
[0054] Both neurons of CNS and PNS may be used as the cells for the
above activity measurements. As described in the section
"BACKGROUND ART", CNS in adult mammals naturally contains a large
amount of regeneration (outgrowth) inhibitor. It is, therefore,
extremely difficult to measure in vivo an inhibitory effect on
neurite outgrowth of CNS-neuron, and such inhibitory effect is
usually measured by an in vitro method as described above. Since
these in vitro methods each have individual characteristics, it is
preferred to use more than one method to confirm the activity.
Although preferred neurons used for a measurement of the activity
are CNS-neurons such as motor neurons in spinal cord or motor
cortex, PNS-neurons in superior cervical ganglion and dorsal root
ganglion may also be used because NI35/250 known as a CNS-neuron
regeneration inhibitor has proved to have effects such as
neurite-growth inhibition and growth-cone collapse activities also
on such PNS-neurons (J. Cell Biol., 106, 1281-1288 (1988); Science,
259, 80-83 (1993)).
[0055] Specific examples of the modified proteins of this
embodiment are described below.
[0056] Based on the structural comparison of known Semaphorins,
most of the conserved amino acids are located in the semaphorin
domain, suggesting that these conserved amino acids are essential
for expression of the activity of Semaphorins. Furthermore, the
present inventors have found that a modified Sema III protein in
which aspartic acid residue at position 198 in its semaphorin
domain has been substituted with glycine did not have the
growth-cone collapse activity (see Reference example 1 below).
Accordingly, the aspartic acid at position 198 of Sema III is
believed essential for expression of the activity. The amino acid
residues corresponding to this position are highly conserved in
known Semaphorins, and they are all aspartic acid with a few
exceptions in which glutamic acid is located at this position. It
is, therefore, believed that the amino acid residue at this
position is also essential for expression of the activity of
Semaphorins other than Sema III. In Semaphorin Y of the present
invention, the amino acid residue corresponding to the position 198
of Sema III is presumed to be aspartic acid at position 197 in the
amino acid sequence shown in SEQ ID NO: 3, or aspartic acid at
position 198 in the amino acid sequence of human Semaphorin Y shown
in SEQ ID NO: 6.
[0057] Considering the above information, it is desirable to make
the above-described deletions, substitutions and/or additions of
amino acids at positions other than those conserved among
Semaphorins, so as to retain the activity of Semaphorin Y in
modified proteins. Particularly, it is desirable not to modify the
aspartic acid at position 197 in rat Semaphorin Y shown in SEQ ID
NO: 3 and the aspartic acid at position 198 in human Semaphorin Y.
In order to substitute amino acids conserved among Semaphorins
while retaining the activity of Semaphorin Y, it is desirable to
substitute an amino acid having a similar side chain for the amino
acid residue to be substituted. By substituting such amino acid
having a similar side chain for a conserved amino acid, it may be
possible to produce a modified protein which has an enhanced
activity of Semaphorin Y. Such modified protein having the enhanced
activity is highly suitable as a neurite-outgrowth inhibitor for
PNS-neuron as will be described hereinafter in the section of the
22nd embodiment of the present invention.
[0058] In the above-noted embodiment, "a conserved amino acid"
refers to an amino acid located at a position at which more than
50% of Semaphorin genes shown in FIG. 2 of Cell, 75, 1389-1399
(1993) or FIG. 1 of Neuron, 14, 941-948 (1995) share the same amino
acid.
[0059] 3) DNA Hybridizing Under Stringent Conditions To Semaphorin
Y Gene Of the above-mentioned DNAs, "a gene which hybridizes under
stringent conditions to Semaphorin Y gene and which encodes a
protein inhibiting neurite outgrowth" refers to a gene such as
Semaphorin Y gene derived from a mammal, which hybridizes under
stringent conditions to rat or human Semaphorin Y gene comprising
the base sequence shown in SEQ ID NO: 1, 2, 4, or 5.
[0060] As used herein, "a gene which hybridizes under stringent
conditions" refers to such a gene that hybridizes to rat or human
Semaphorin Y gene, for example, when subjected to hybridization at
a formamide concentration of about 45% (v/v) and a salt
concentration of about 5.times. SSPE and at a temperature around
42.degree. C., and washed at a salt concentration of about 2.times.
SSPE and at a temperature around 42.degree. C. Cloning of such
genes may be achieved, for example, by screening cDNA or genomic
libraries prepared from various animal tissues using all or part of
DNA shown in SEQ ID NO: 1 or 4 as a probe. Such screening may be
carried out by making reference to the standard texts such as
"Molecular Cloning 2nd ed." (Cold Spring Harbor Laboratory Press
(1989)).
[0061] Specific examples of the gene of this embodiment may include
all the Semaphorin Y genes of mammal and avian. Between mammals or
between mammal and avian, homologous genes have quite similar
sequences, and usually more than 80%, in many cases more than 90%,
of the base sequence are common to each other. All the mammal and
avian Semaphorin Y genes, therefore, correspond to this embodiment.
In other words, those genes which have a homology of 80% or above,
and preferably of 90% or above, are included in this
embodiment.
[0062] The 3rd embodiment of the present invention is a gene
comprising DNA which hybridizes under stringent conditions to DNA
comprising the base sequence shown in SEQ ID NO: 7, and which
encodes a protein having a semaphorin domain.
[0063] In the above description, "DNA comprising the base sequence
shown in SEQ ID NO: 7" refers to a fragment cloned by PCR using the
sequence information of the DNA "R59527" which encodes, in part, a
sequence consisting of seven amino acids well conserved among
Semaphorins (Gln (or Arg)-Asp-Pro-Tyr-Cys-Ala (or Gly)-Trp), and
the DNA fragment corresponds to a region from position 1574 to
position 1743 in the base sequence of rat Semaphorin Y shown in SEQ
ID NO: 1, or a region from position 1524 to position 1693 in the
base sequence of human Semaphorin Y shown in SEQ ID NO: 4.
[0064] The "stringent conditions" refers to those conditions
described above in the section of the 2nd embodiment of the present
invention.
[0065] Cloning of these DNAs is achieved by, for example,
hybridization with DNA of SEQ ID NO: 7, and specifically may be
carried out, for example, according to the procedures described in
TINS, 15, 319-323 (1992) and references cited therein, and more
specifically according to the following procedures.
[0066] That is, the cloning may be achieved by screening cDNA or
genomic libraries prepared from various animal tissues using DNA
consisting of the base sequence shown in SEQ ID NO: 7 as a probe.
The screening may be carried out according to, for example, the
procedures as described in Example 1. Preferred cDNA libraries are
those derived from an adult tissue of CNS, and cDNA libraries
derived from hippocampus, corpus striatum, and cerebellum are more
preferred. As described above, the conditions shown in Example 1 or
those described in TINS, 15, 319-323 (1992) and references cited
therein may be used for the hybridization.
[0067] The DNA of this embodiment is also "DNA which encodes a
protein having a semaphorin domain". As used herein, "semaphorin
domain" refers to a domain consisting of 300-600 amino acid
residues more than 20% of which are identical to those amino acids
constituting the semaphorin domain of any one of ten known
Semaphorins (G-Sema I, T-Sema, I, D-Sema II, H-Sema III,
C-Collapsin, Sem A, Sem B, Sem C, Sem D, Sem E) described in, for
example, Cell, 75, 1389-1399 (1993) or Neuron, 14, 941-948 (1995).
Those proteins having a semaphorin domain more than 30% of which
amino acids are identical to those amino acids in any one of the
known Semaphorins are particularly preferred. The identity of amino
acids is determined by comparison using, for example, DNASIS Ver.
2.0 (HITACH Software Engineering) under conditions of ktup=1 and
cutoff=1. More preferred proteins are those in which ten or more
cysteines, particularly twelve or more cysteines, of the thirteen
cysteines conserved in semaphorin domains of the ten known
Semaphorins (for example, those cysteines marked in FIG. 1 on page
942 of Neuron, 14, 941-948 (1995)) are conserved.
[0068] Examples of such gene of this embodiment may include
Semaphorin genes which hybridize under stringent conditions to DNA
comprising the base sequence shown in SEQ ID NO: 7 and which
contain semaphorin domains and exhibit the neurite-outgrowth
inhibition activity, including all of the Semaphorin Y genes of
mammal and avian.
[0069] The 4th embodiment of the present invention is a protein
obtained by expressing the gene of any one of the above items (1)
to (3).
[0070] Typical examples of protein included in this embodiment are
rat Semaphorin Y comprising the amino acid sequence shown in SEQ ID
NO: 3, and human Semaphorin Y comprising the amino acid sequence
shown in SEQ ID NO: 6. The rat or human Semaphorin Y contains a
signal sequence at its N-terminus and such signal sequence is
presumed to correspond to a region from position 1 to position 23
of the amino acid sequence shown in SEQ ID NO: 3 or from position 1
to position 24 of the amino acid sequence shown in SEQ ID NO: 6,
respectively. Since the signal sequence is removed by processing
during its transfer to membrane, such mature forms of Semaphorin Y
are also included in this embodiment.
[0071] Preparation of the proteins of this embodiment may be
achieved, for example, by ligating a cloned rat Semaphorin Y cDNA
into a known expression vector such as pET or pCDM8, and
introducing it into appropriate host cells to express and produce
Semaphorin Y. The host cells may be prokaryotic or eukaryotic. For
example, Escherichia coli strains or animal cell lines are already
conventionally used for such purpose and are commercially or
publicly available. Examples of animal host cells include COS-1,
COS-7, CHO cells and the like.
[0072] To transform appropriate animal host cells with an
expression plasmid, a known procedure such as DEAE-dextran method
(Current Protocols in Molecular Biology, F. M. Ausubel et al. ed.,
John Wiley & Sons (1987)) may be used. As confirmed in Example
6, Semaphorin Y exists in the cell membrane faction which contains
a sufficient amount of Semaphorin Y to be directly used in various
assays. Therefore, various assays for activities of a protein of
this embodiment may easily be conducted using a cell membrane
fraction prepared from appropriate cells.
[0073] Furthermore, a protein of this embodiment may be purified
by, for example, affinity purification using Semaphorin
Y-recognizing antibodies described hereinafter in the section of
the 16th embodiment of the present invention, or conventional
column chromatography.
[0074] The 5th embodiment of the present invention is a gene
encoding a protein which comprises an amino acid sequence wherein
one or more amino acids are deleted, substituted and/or added in
the rat or human Semaphorin Y shown in SEQ ID NO: 3 or 6 and which
protein promotes neurite outgrowth. The 6th embodiment of the
present invention is a protein obtained by expressing the gene of
the 5th embodiment of the present invention.
[0075] In the genes of the above 5th embodiments, deletions,
substitutions and/or additions may be introduced in the procedures
similar to those used for a gene encoding a modified protein of the
1st embodiment of the present invention. Similarly, the promotion
effect on neurite outgrowth may easily be measured, for example, by
adding Semaphorin Y in an assay system for Semaphorin Y activity
described above in the section of the 1st embodiment of the present
invention and further adding thereto a test substance (i.e., a
candidate modified Semaphorin Y protein). For details, see the
descriptions in the section of the 18th embodiment of the present
invention.
[0076] Specific examples of the proteins of the 6th embodiment may
be modified proteins of which neurite-outgrowth inhibition activity
has been eliminated. Such modified protein lacking the
neurite-outgrowth inhibition activity is expected to exert the
promotion effect on neurite-outgrowth, when it binds to receptors
for Semaphorin Y or to Semaphorin Y itself, by inhibiting the
binding of Semaphorin Y to the receptors. As described above in the
section of the 1st embodiment of the present invention, it has been
suggested that the active site of Semaphorin may be located in the
semaphorin domain, and particularly, it may be located at aspartic
acid at position 197 in rat Semaphorin Y or aspartic acid at
position 198 in human Semaphorin Y. Accordingly, in order to
eliminate the semaphorin Y activity from the modified protein, it
is desirable to introduce the deletions, substitutions and/or
additions to the conserved amino acids in said semaphorin domain,
preferably to the aspartic acid at position 197 in rat Semaphorin Y
or to the aspartic acid at position 198 in human Semaphorin Y. In
such cases, those substitutions in which an amino acid having a
side chain of a distinct nature is substituted for the original
amino acid are desirable. Also in the cases of Semaphorin Y other
than that from human or rat, modifications are preferably made on
aspartic acid at this position, that is, on amino acid residue at
the position which corresponds to position 197 in rat Semaphorin Y
or to position 198 in human Semaphorin Y when the amino acid
sequence of said Semaphorin Y is aligned with that of rat or human
Semaphorin Y so as to give the maximum identity.
[0077] Since the proteins of the 6th embodiment of the present
invention promote neurite outgrowth as described above, some of
these proteins will serve as CNS-neuron regeneration promoters as
described hereinafter in the section of the 21st embodiment.
[0078] The 7th embodiment of the present invention is DNA which is
cloned from a human cDNA library or a human genomic library, and
which hybridizes under stringent conditions to DNA comprising at
least part of rat or human Semaphorin Y DNA shown in SEQ ID NO: 1
or 4, respectively.
[0079] Methods of cloning are described in detail in, for example,
"Molecular Cloning 2nd ed.", Cold Spring Harbor Laboratory Press
(1989), and specifically include, for example, methods employing
hybridization or PCR. Although a preferred library used herein is a
genomic library derived from human, a cDNA library derived from
CNS-neuron in the adult may also be used. Those methods employing
hybridization may be carried out according to, for example, TINS,
15, 319-323 (1992) and references cited therein. Those methods
employing PCR may be carried out according to, for example, "PCR"
edited by McPherson et al., IRL Press (1991).
[0080] The DNAs thus cloned include not only the full length DNA
but also its DNA fragments comprising more than 200 bases, or
single-stranded forms (coding strands or complementary stands
thereof) of said DNA fragments. Specific examples of DNA of the 7th
embodiment of the present invention may include chromosomal DNAs
containing 5' and/or 3' transcriptional control regions, noncoding
sequences of exons, introns or the like, in addition to regions
encoding amino acids. Such sequences which do not encode any amino
acids are also quite useful, for example, in developing a medicine
using antisense techniques described hereinafter.
[0081] The 8th embodiment of the present invention is an expression
plasmid which expresses either the gene of the 1st, 2nd, 3rd or 5th
embodiment, or DNA of the 7th embodiment of the present invention.
The 9th embodiment of the present invention is a transformant
transformed with the expression plasmid of the 8th embodiment.
Furthermore, the 10th embodiment of the present invention is a
process for producing a recombinant protein which process comprises
culturing the transformant of the 9th embodiment and recovering the
recombinant protein expressed. As described above in the section of
the 4th embodiment of the present invention, methods of preparing
an expression plasmid and a transformant, and methods of producing
a recombinant protein, per se, are all well known to those skilled
in the art.
[0082] The 11th embodiment of the present invention is a peptide
comprising at least 6 amino acids of a protein of the 4th or 6th
embodiment of the present invention. In this context, the
limitation "at least 6 amino acids" is based on the fact that a
minimal size of peptide capable of forming a stable structure
consists of 6 amino acids, and preferred peptides are those
consisting of 8 or more amino acids, more preferably of about 10-20
amino acids. A short peptide such as those consisting of about
10-20 amino acids can be synthesized on a peptide synthesizer,
while a longer peptide can be obtained by preparing DNA through
conventional genetic engineering, and expressing it in, for
example, animal cells as described above. The peptide thus prepared
can also be modified by conventional methods.
[0083] These peptides can be applied to pharmaceutical agents
described hereinafter in the section of the 12th and 13th
embodiments, and can also be used for producing antibodies.
[0084] The 12th embodiment of the present invention is a peptide of
the 11th embodiment of the present invention which promotes neurite
outgrowth. Such polypeptide may be prepared by the methods
described above in the section of the 11th embodiment of the
present invention. The promotion effect on neurite outgrowth can
also easily be measured as described above in the section of the
5th embodiment of the present invention by adding Semaphorin Y to
an activity assay system described above in the section of the 1st
embodiment of the present invention and further adding thereto a
test substance (i.e., a candidate peptide of Semaphorin Y). For
details, see the descriptions in the section of the 18th embodiment
of the present invention.
[0085] Specific examples of these peptides may be peptides which
have lost the neurite-outgrowth inhibition activity of Semaphorin
Y. A peptide lacking Semaphorin Y activity is expected to exert its
neurite-outgrowth promotion effect, when it binds to receptors for
Semaphorin Y or to Semaphorin Y itself, by inhibiting the binding
of Semaphorin Y to the receptors. Some of such peptides will serve
as CNS-neuron regeneration promoters as described hereinafter in
the section of the 21st embodiment.
[0086] The 13th embodiment of the present invention is a peptide of
the 11th embodiment of the present invention, characterized in that
it contains the aspartic acid residue at position 198 of the amino
acid sequence shown in SEQ ID NO: 6 or an amino acid residue
corresponding to the position of said aspartic acid residue. Such
peptides may be prepared by the methods described above in the
section of the 11th embodiment.
[0087] As described above in the section of the 1st embodiment of
the present inventions, the aspartic acid residue at position 198
of human Semaphorin Y shown in SEQ ID NO: 6 (in the case of rat,
the aspartic acid residue at position 197) seems essential for
expression of the activity of Semaphorin Y. Since this amino acid
residue may possibly be involved in the binding between Semaphorin
Y and its receptors, a peptide of this embodiment containing this
amino acid residue may interfere the neurite-outgrowth inhibition
activity of Semaphorin Y by binding to receptors for Semaphorin Y
or to Semaphorin Y itself, resulting in promotion of neurite
outgrowth. Some of the peptides having such effect will serve as
CNS-neuron regeneration promoters as described hereinafter in the
section of the 21st embodiment. Such neurite-outgrowth promotion
activity can easily be measured as described above in the section
of the 5th embodiment of the present invention by adding Semaphorin
Y to an activity assay system described in the section of the 1st
embodiment of the present invention, and further adding thereto a
test substance (i.e., a candidate peptide of Semaphorin Y). For
details, see the descriptions in the section of the 18th embodiment
of the present invention.
[0088] In this embodiment, "an amino acid corresponding to the
position of said aspartic acid" refers to an amino acid residue
which is located at the position corresponding to position 198 in
human Semaphorin Y, when the amino acid sequence of the protein of
the 4th or 6th embodiment, of the present invention is aligned with
the amino acid sequence of human Semaphorin Y shown in SEQ ID NO: 6
so as to give the maximum identity. Accordingly, "a peptide
characterized in that it contains an amino acid corresponding to
the position of said aspartic acid" refers to a peptide which
comprises such amino acid at the position corresponding to position
198 in human Semaphorin Y as well as flanking amino acids on either
side thereof.
[0089] The 14th embodiment of the present invention is an antisense
nucleotide, or chemically modified variant thereof, which is
directed against a segment comprising at least eight or more bases
in the gene of any one of the 1st to 3rd embodiments, or in DNA of
the 7th embodiment of the present invention.
[0090] As used herein, "antisense nucleotide" refers to a so-called
antisense oligonucleotide, antisense RNA, or antisense DNA, and it
may be artificially prepared using a DNA synthesizer, or may be
obtained by, for example, expressing a gene in the direction
opposite to the usual case (i.e., in the antisense direction). For
details, see the descriptions in the section of the 21st embodiment
of the present invention.
[0091] These antisense nucleotides are used for inhibiting the
expression of Semaphorin Y as described hereinafter in the section
of the 15th embodiment of the present invention, and are also
useful as laboratory reagents for, for instance, in situ
hybridization. In the present invention, "a chemically modified
variant" specifically refers to such a variant that is chemically
modified so as to enhance the transferability of the antisense
nucleotide into cells or the stability of the antisense nucleotide
in the cells. Examples of such chemically modified variant are
phosphorothioate, phosphorodithioate, alkylphosphotriester, alkyl
phosphonate, alkyl phosphoamidate and the like derivatives
("Antisense RNA and DNA", WILEY-LISS, 1992, pp. 1-50, J. Med.
Chem., 36, 1923-1937 (1993)). The chemically modified variant may
be prepared according to, for example, the references cited just
above.
[0092] The 15th embodiment of the present invention is an antisense
nucleotide, or chemically modified variant thereof, of the 14th
embodiment described above, characterized in that it inhibits the
expression of the protein of the 4th embodiment of the present
invention.
[0093] mRNAs produced by usual gene transcription are
sense-strands, and the antisense nucleotides or chemically modified
variants thereof can bind to such sense-strand mRNAs in cells to
inhibit the expression of those particular genes. Therefore, the
above-described antisense nucleotides or chemically modified
variants thereof can inhibit the expression of Semaphorin Y, and
can thereby inhibit the activity of Semaphorin Y. Some of antisense
nucleotides or chemically modified variants thereof having such
effect will serve as CNS-neuron regeneration promoters as described
hereinafter in the section of the 21st embodiment of the present
invention.
[0094] It can easily be determined whether a particular antisense
nucleotide prepared, or a chemically modified variant thereof, has
a desired inhibitory effect or not, by directly introducing the
antisense oligonucleotide itself or by introducing a gene which
produces said antisense RNA when transcribed, into cells expressing
Semaphorin Y, and then determining whether the amount of the
expressed Semaphorin Y is decreased or not.
[0095] Examples of antisense nucleotide having such inhibitory
effect are those oligonucleotides having sequences complementary to
either the coding region or the 5' noncoding region of Semaphorin
gene of the above-described embodiments. Especially preferred are
those antisense nucleotides having sequences complementary to the
transcription initiation site, translation initiation site, 5'
noncoding region, exon-intron junction region, or 5' CAP
region.
[0096] The 16th embodiment of the present invention is an antibody
against the protein of the 4th or 6th embodiment, or against the
peptide of any one of the 11th to 13th embodiments. Such antibody
can easily be produced by using mouse or rabbit according to the
procedures described in, for example, "Current Protocols in
Immunology", pp. 2.4.1-2.6.6 (1992, J. E. Coligan ed.). Monoclonal
antibodies can also easily be produced by the methods described in
the above-mentioned reference. Such antibodies may be used in
affinity chromatography or screening of cDNA libraries, and as
pharmaceutical or diagnostic agents, or laboratory reagents. Some
of such antibodies have the activity of neutralizing Semaphorin Y.
Such neutralizing activity can easily be determined, as described
above in the section of the 5th embodiment of the present
invention, by adding Semaphorin Y to an activity assay system
described in the section of the 1st embodiment of the present
invention, and further adding thereto a test substance (i.e., a
candidate antibody against Semaphorin Y). Some of such neutralizing
antibodies will serve as CNS-neuron regeneration promoters as
described hereinafter in the section of the 21st embodiment of the
present invention.
[0097] The 17th embodiment of the present invention is a
pharmaceutical agent comprising, as an active ingredient, any one
of all of the genes (DNAs), proteins, peptides, antisense
nucleotides or chemically modified variants thereof, and antibodies
of the present invention.
[0098] Among such pharmaceutical agents, CNS-neuron regenerators
and neurite-outgrowth inhibitors for PNS-neuron will be described
in the sections of the 21st and 22nd embodiments of the present
invention, respectively. See, therefore, the sections of the 21st
and 22nd embodiments for such applications.
[0099] It is being demonstrated in recent years that certain
Semaphorins play important roles not only in the nervous system but
also in non-nervous system. For example, it has been suggested that
Semaphorin may probably act in inhibiting the growth of cardiac
muscles (Nature, 383, 525-528 (1996)). Also in the immune system,
certain Semaphorin has been suggested to be involved in aggregation
and survival of B lymphocytes (Proc. Natl. Acad. Sci. USA, 93,
11780-11785 (1996)). It has also been suggested more recently that
a certain Semaphorin may play some role in the immune reactions in
rheumatism (B.B.R.C., 234, 153-156 (1997)). Furthermore,
involvement of Semaphorins in lung cancer has also been suggested
(Proc. Natl. Acad. Sci. USA, 93, 4120-4125 (1996)).
[0100] Accordingly, Semaphorin Y of the present invention or its
modified proteins, peptides, antisense nucleotides and the like are
expected to be useful as antiallergic agents, immunosuppressive
agents, or anti-tumor agents. For specific directions for use,
dosage and the like, see the sections of the 21st and 22nd
embodiments.
[0101] The 18th embodiment of the present invention is a method of
screening for Semaphorin Y antagonists, characterized in that it
employs the protein of the 4th embodiment of the present invention.
As used herein, "Semaphorin Y antagonist" refers to a substance
which inhibits, for example, the neurite-outgrowth inhibition
activity of Semaphorin Y.
[0102] The screening is conducted by adding Semaphorin Y to an
assay system for Semaphorin Y activity described in the section of
the 1st embodiment of the present invention, and further adding
thereto a test substance. In particular, inhibition of the
Semaphorin Y activity resulted from the addition of the test
substance to the culture medium throughout the incubation period or
only temporarily in the incubation period can be used as an
indicator in the Semaphorin Y activity assay conducted with added
Semaphorin Y. It is also important to confirm that the test
substance alone does not influence the survival and
neurite-outgrowth of neurons at the same concentration. When both
of these requirements are fulfilled, one can consider the test
substance as a Semaphorin Y antagonist. Although it is preferred to
prepare in advance the test substance in the form of aqueous
solution, an organic solvent such as DMSO may also be used as a
solvent. In any cases, it is important to minimize the volume of
solvent so as to exclude any effects of the solvent on neurons.
Specifically, the volume to be added should be less than an equal
volume, preferably less than {fraction (1/10)} volume, and more
preferably less than {fraction (1/100)} volume relative to the
culture medium. Some of Semaphorin Y antagonists thus obtained will
serve as CNS-neuron regeneration promoters as described hereinafter
in the section of the 21st embodiment of the present invention.
[0103] The 19th embodiment of the present invention is Semaphorin Y
antagonist obtained by the screening method of the 18th embodiment
of the present invention. Such antagonist may have any structure
and any form, provided that it inhibits the activity of Semaphorin
Y.
[0104] The 20th embodiment of the present invention is Semaphorin Y
antagonist of the 19th embodiment which comprises the protein of
the 6th embodiment, the peptide of any one of the 11th to 13th
embodiments, or the antibody of the 16th embodiment of the present
invention. In other words, it is a protein of the 6th embodiment, a
polypeptide of any one of the 11th to 13th embodiments, or an
antibody of the 16th embodiment of the present invention which has
an effect of inhibiting the activity of Semaphorin Y. Such
antagonists can be identified by subjecting the above substances to
the screening system of the 18th embodiment of the present
invention, and some of the antagonists thus identified will serve
as CNS-neuron regeneration promoters as described below in the
section of the 21st embodiment of the present invention.
[0105] The 21st embodiment of the present invention is a CNS-neuron
regeneration promoter, characterized in that it contains at least
one of the antisense nucleotides or chemically modified variants
thereof of the 14th or 15th embodiment, or Semaphorin Y antagonists
of the 19th or 20th embodiment of the present invention. Since this
embodiment relates to the use of substances in "regeneration
therapy for CNS-neuron", specific directions for use, dose and the
like, of the substances are described below.
[0106] 1) Antisense nucleotide or chemically modified variant
thereof Application of antisense nucleotides has been attempted in
various diseases, and in recent years, it is also considered to be
applicable in neurological disorders (TINS 20, No. 8, 321-322
(1997)).
[0107] As described above in the section of the 14th or 15th
embodiment of the present invention, the antisense nucleotide or
chemically modified variant thereof of the 14th or 15th embodiment
of the present invention can be used for inhibiting expression of
Semaphorin Y gene. Accordingly, such antisense nucleotide may
decrease the abundance of the Semaphorin protein, and promote
regeneration of CNS-neurons. Therapeutic methods using the
nucleotide or the variant include those in which the antisense
oligonucleotide or its chemically modified variant itself is
administered, and those in which antisense RNA is produced in
cells.
[0108] In the method in which the antisense oligonucleotide or its
chemically modified variant is administered as such, a preferred
antisense oligonucleotide has a length, for example, about 5-200
bases, more preferably 8-25 bases, and especially preferably 12-25
bases. Antisense oligonucleotide or its chemically modified variant
may be formulated by mixing it with stabilizing agent, buffer,
solvent and the like prior to its administration. Such formulation
may be co-administered with, for example, an antibiotic,
anti-inflammatory, or anesthetic agent. Although the formulation
thus prepared may be administered via various routes, it is
preferred to topically administered at a site in which neurons are
notably disordered. Usually, regeneration of neuron takes several
days to several months, and the formulation is administered every
day or every several days to several weeks during the period. To
avoid such frequent administrations, a sustained-release
mini-pellet formulation may be prepared and embedded near the
affected site. Alternatively, a formulation may be gradually and
continuously administered to the affected site by means of, for
example, an osmotic pump. The dose is typically adjusted so that
the concentration at the site of action will be 0.1 nM to 10
.mu.M.
[0109] In the method in which antisense RNA is produced in cells, a
preferred antisense RNA has a length of, for example, more than 100
bases, preferably more than 300 bases, and more preferably 500
bases or more.
[0110] The methods by which a gene expressing an antisense RNA is
introduced into a patient include an in vivo method in which the
gene is directly introduced into cells in a living body, and an ex
vivo method in which the gene is introduced into particular cells
ex vivo and the cells are returned into the body (Nikkei Science,
April, 1994, pp. 20-45; Gekkan-Yakuji, 36 (1), 23-48 (1994);
Jikkenn-Igaku-Zokan, 15 (15), 1994; and references cited therein).
An in vivo method is more preferred.
[0111] Such in vivo methods include a method employing recombinant
viruses and other methods (Nikkei Science, April, 1994, pp. 20-45;
Gekkan-Yakuji, 36 (1), 23-48 (1994); Jikken-Igaku-Zokan, 12 (15),
in its entirety (1994); and references cited therein).
[0112] The methods employing recombinant viruses may include the
methods in which Semaphorin gene is incorporated into a virus
genome of, for example, retrovirus, adenovirus, adeno-associated
virus, herpesvirus, vaccinia virus, poliovirus, or sindbis virus,
and the recombinant virus is introduced into a living body. Among
these methods, those employing retrovirus, adenovirus or
adeno-associated virus are particularly preferred.
[0113] Other methods may include a liposome method or a lipofectin
method. The liposome method is particularly preferred.
[0114] For the ex vivo methods, a micro-injection method, the
calcium phosphate method, electroporation and the like may also be
used, besides those techniques described above.
[0115] Administration of the gene to a patient is carried out via
appropriate routes depending on particular disease or symptom to be
treated, and the like. For example, it may be administered
intravenously, intraarterially, subcutaneously, or intramuscularly,
or directly administered into an affected site such as neuron. For
example, when spinal cord is infected with the recombinant viruses,
the expression of Semaphorin gene is inhibited exclusively in the
spinal cord. Expression of antisense oligonucleotide of the present
invention typically lasts several days to several months, and such
single infection is sufficient to allow regeneration of neuron. The
gene may also be re-infected, when weakly expressed. When
administered by an in vivo method, the gene may be formulated in
the form of, for example, a solution, and typically it is
formulated in the form of an injection containing Semaphorin gene
as an active ingredient to which conventional carrier and the like
may be added, if necessary. In the case of liposomes or
membrane-fused liposomes (such as Sendai virus (HVJ)-liposomes)
containing Semaphorin gene, the liposome preparations may be in the
form of a suspension, a frozen preparation, a
centrifugally-concentrated frozen preparation or the like.
[0116] Although the amount of Semaphorin gene in the formulation
may vary depending on the disease to be treated, the age and weight
of the patient, and the like, it is typically 0.0001-100 mg, and
preferably 0.001-10 mg, and such formulation is preferably
administered once every several days to several months.
[0117] 2) Modified protein of Semaphorin Y
[0118] As described above in the sections of the 5th and 6th
embodiments of the present invention, one can prepare a modified
Semaphorin Y of which neurite-outgrowth inhibition activity on
CNS-neuron has been eliminated. When administered into a living
body, such modified protein is expected to bind to receptors for
Semaphorin Y in place of Semaphorin Y, resulting in inhibition of
Semaphorin Y activity and promotion of regeneration of
CNS-neuron.
[0119] Such modified protein of Semaphorin Y is formulated with
stabilizer, buffer, and diluent, and administered to a patient for
therapy. Such formulation may be administered via various routes,
and it is preferred to topically administer to the focal site.
Since regeneration of neuron typically takes several days to
several months, the formulation is administered once or more in
order to continuously inhibit Semaphorin Y activity throughout the
period. When administered more than once, it is desirable to
administer it every day or repeatedly at appropriate intervals.
When administered to CNS by injection, for example, into spinal
cord, several hundreds .mu.g to 2 g, preferably less than several
tens mg, are used per administration. To reduce the administration
frequency, it may be administered using a sustained-release
formulation or gradually administered over a long period by means
of, for example, an osmotic pump. Alternatively, it may be
administered by grafting cells expressing such modified Semaphorin
Y protein into a living body.
[0120] 3) Peptide of Semaphorin Y
[0121] Some of the peptides of any one of the embodiments from 11th
to 13th of the present invention suppress the neurite outgrowth
inhibition activity of Semaphorin Y on CNS-neuron by inhibiting the
binding of Semaphorin Y to its receptors, resulting in promotion of
CNS-neuron regeneration. Examples of peptide having such effect
include a peptide characterized in that it contains aspartic acid
residue at position 198 of human Semaphorin Y shown in SEQ ID NO: 6
or an amino acid residue at the position corresponding to that of
said aspartic acid residue, as described above in the section of
the 13th embodiment of the present invention. The suppression may
be any one of competitive, noncompetitive, uncompetitive, and
allosteric inhibitions.
[0122] As for the methods of formulating or administering such
polypeptides, and their doses, see the above section "2) Modified
protein of Semaphorin Y".
[0123] 4) Antibody against Semaphorin Y
[0124] A neutralizing antibody which neutralizes the activity of
Semaphorin Y is expected to promote the regeneration therapy of
CNS-neuron by inhibiting Semaphorin Y activity, when administered
into a living body.
[0125] The methods of formulating or administering such
neutralizing antibodies and their doses may be the same as
described in the above section "2) Modified protein of Semaphorin
Y". Alternatively, a method in which cells producing a monoclonal
antibody are grafted directly into CNS may also be used, as
described in Nature, 343, 269-272 (1990).
[0126] The 22nd embodiment of the present invention is a neurite
outgrowth inhibitor for PNS-neuron, characterized in that it
contains at least one of the proteins of the 4th embodiment of the
present invention. Although the proteins of the 4th embodiment of
the present invention may inhibit the neurite outgrowth of
CNS-neuron, they are also expected to inhibit the neurite outgrowth
of PNS-neuron, since PNS-neuron also probably expresses receptors
for Semaphorin Y, and receptors for other Semaphorins also probably
react with Semaphorin Y. Accordingly, they may serve as therapeutic
agents for atopic dermatitis, pain or other diseases by virtue of
their inhibition activity on neurite outgrowth of PNS-neuron.
[0127] As for the methods of formulating or administering such
proteins, and their doses, see the above section "2) Modified
protein of Semaphorin Y".
[0128] The 23rd embodiment of the present invention is a transgenic
animal in which either the gene of any one of the 1st to 3rd and
5th embodiments, or DNA of the 7th embodiment of the present
invention has been artificially inserted into its chromosome, or
has been knocked out.
[0129] As apparent from the following references, one skilled in
the art can quite easily produce a transgenic animal which
expresses the gene of the 1st, 4th, 7th, or 9th embodiment of the
present invention, in the light of the gene information on
Semaphorin Y of the present invention: "Manipulation of Mouse
Embryo" edited by B. Hogan et al., 1986, Cold Spring Harbor
Laboratory; Shinichi Aizawa, "Gene Targeting", 1995, Yodosha, etc.
Accordingly, the transgenic animal thus produced is naturally
included within the scope of the present invention. The transgenic
animal thus produced is very useful as an animal model for
developing pharmaceuticals or as an animal used for screening of
pharmaceuticals. Furthermore, a so-called knockout animal in which
the gene of the 1st, 4th, 7th, or 9th embodiment of the present
invention has been deleted is characterized in that it does not
contain such gene. As described in literatures, or as apparent from
the common knowledge in the art, such knockout animals cannot be
produced without the gene information on Semaphorin Y of the
present invention. It goes without saying, therefore, that such
knockout animals are included within the scope of the present
invention.
[0130] While Semaphorin Y has an important in vivo function
relating to regeneration of neurons as described above, it has been
also suggested as mentioned above that Semaphorin Y may have other
unknown functions such as immunosuppression (Cell, 75, 1389-1399
(1993)). Accordingly, it is quite important to investigate the
expression of Semaphorin Y gene or the distribution and function of
Semaphorin Y protein for studying this technical field or for
diagnosing patients with neurological disorders or other diseases.
The present invention can also provide gene probes, antibodies,
recombinant proteins, transgenic animals and the like which can be
used for such purposes.
BRIEF DESCRIPTION OF DRAWINGS
[0131] FIG. 1 shows a picture of electrophoresis indicating
distribution of Semaphorin Y expression among various tissues
determined by Northern analysis.
[0132] Total RNAs were extracted from various tissues of six-weeks
old rats, electrophoresed on 1% agarose-formamide gel, blotted onto
a filter, and hybridized with a .sup.32P-labeled rat Semaphorin Y
DNA probe to determine the distribution of Semaphorin Y mRNA
expression. Fifteen .mu.g of RNA was loaded in each lane. The upper
panel shows the result of autoradiography. The positions
corresponding to 18S and 28S ribosomal RNAs are indicated at the
left margin of the panel. The lower panel shows the ethidium
bromide staining of the gel. The upper and lower bands correspond
28 and 18S ribosomal RNAs, respectively.
[0133] FIG. 2 shows a picture of electrophoresis indicating
distribution of Semaphorin Y expression among CNS tissues
determined by Northern analysis.
[0134] Total RNAs were extracted from CNS tissues of six-weeks old
rats, electrophoresed on 1% agarose-formamide gel, blotted onto a
filter, and hybridized with a .sup.32P-labeled rat Semaphorin Y DNA
probe to determine the distribution of Semaphorin Y mRNA
expression. Fifteen .mu.g of RNA was loaded in each lane. The upper
panel shows the result of autoradiography. The positions
corresponding to 18S and 28S ribosomal RNAs are indicated at the
left margin of the panel. The lower panel shows the ethidium
bromide staining of the gel. The upper and lower bands correspond
28 and 18S ribosomal RNAs, respectively.
[0135] FIG. 3 shows a picture of electrophoresis indicating
distribution of Semaphorin Y mRNA expression among human CNS
tissues determined by Northern analysis.
[0136] A Membrane filter onto which mRNAs prepared from various
regions of human CNS tissues have been transferred after being
electrophoresed (2 .mu.g/lane) (Clontech) was hybridized with
.sup.32P-labeled Semaphorin Y DNA probe to determine the
distribution of Semaphorin Y mRNA expression. The figure shows the
result of autoradiography. In this figure, the arrows indicate the
positions of Semaphorin Y mRNA bands. Positions of size makers are
indicated in kb at the left margin of the figure.
[0137] FIG. 4 shows a picture of electrophoresis indicating
expression of Semaphorin Y protein in COS 7 cells.
[0138] An expression plasmid for Semaphorin Y having additional 10
amino acids derived from human c-Myc added at its C-terminus was
constructed, and introduced into COS 7 cells for transient
expression (indicated as rSYmyc). A plasmid containing no
Semaphorin Y gene was used as control (indicated as Control). At
day 3 after introducing plasmids, the cells were harvested, and the
membrane fraction was prepared. The membrane fraction was
fractionated by SDS-PAGE, and then subjected to Western blotting
using an anti-Myc antibody. In this figure, the arrow indicates the
position of the band of Semaphorin Y protein having added Myc
peptide. Positions and molecular weights of size makers are
indicated in kD at the left margin of the figure.
[0139] FIG. 5 shows a picture of electrophoresis indicating the in
vivo distribution of Semaphorin III expression among various
tissues determined by Northern analysis.
[0140] Total RNAs were extracted from various tissues of adult
rats, electrophoresed on 1% agarose-formamide gel, blotted onto a
filter, and hybridized with .sup.32P-labeled mouse Semaphorin III
DNA probe to determine the distribution of Semaphorin III mRNA
expression. Fifteen .mu.g of RNA was loaded in each lane. The upper
panel shows the result of autoradiography. The positions of 18S and
28S ribosomal RNAs are indicated at the left margin of the figure.
The lower panel indicates the ethidium bromide staining of the gel.
The upper and lower bands correspond to 28S and 18S ribosomal RNAs,
respectivly.
EXAMPLES
[0141] Fundamental procedures for experiments are described in
detail in many publications such as "Molecular Cloning, 2nd Ed."
edited by Maniatis et al. (Cold Spring Harbor Laboratory Press,
1989), "Current Protocols in Molecular Biology" edited by Ausubel
et al. (John Wiley & Sons, 1987), and "Saibo-Kogaku-Jikken
Protocols" edited by Department of Oncology, The Institute of
Medical Science, The University of Tokyo (Shujunsha, 1991). The
present invention is not intended to be limited by the following
examples, and the examples may be of course modified as usual.
Example 1
Isolation of Rat Semaphorin Y Gene
[0142] (1) Search through Database for a Novel Semaphorin Gene
[0143] Using the dbEST database of the National Center for
Biotechnology Research (Bethesda, Md., US), search was performed
for a sequence which encodes an amino acid sequence relatively well
conserved in known Semaphorin genes and which -is found in only
cDNAs from postnatal brain but not in cDNAs from peripheral
tissues. As a result, the base sequence of File No. R59527 proved
to encode a sequence consisting of seven amino acids common to
known Semaphorin genes (Gln (or Arg)-Asp-Pro-Tyr-Cys-Ala (or
Gly)-Trp). However, the sequence information of R59527 consisting
of 238 bases is so short compared with the cDNAs for known
Semaphorin genes, and only several percent of the total bases could
be translated to a sequence common to those in known Semaphorins.
In addition, the reading frame could not be determined because the
sequence of R59527 is not the one finally determined. It was,
therefore, impossible to conclude that the base sequence of R59527
is part of a novel Semaphorin gene. Then, the inventors firstly
confirmed that a gene containing the above sequence is expressed in
the adult brain, and then sought to clone the full length cDNA
containing the above sequence and determine its gene structure.
[0144] (2) Confirmation of the Expression of the Gene Containing
the Sequence of R59527 in the Brain
[0145] To confirm that the gene is expressed in the adult human
CNS, two DNA primers bounding a segment of about 170 bp, 5'
TGGCTGTATTGTCTACCT 3' (SEQ ID NO: 8) and 5' TGGATTCCTGGTTCCNAGCC 3'
(SEQ ID NO: 9), were synthesized on the basis of the base sequence
of R56527, and used in PCR under conventional conditions together
with cDNAs prepared from a human brain cDNA library (Clontech) as
templates. As a result, about 170 bp fragment was amplified as
expected. The DNA was then cloned into PCRII (Invitrogen) according
to the manufacturer's protocol, and the base sequence was
determined to confirm that the fragment has the same base sequence
as that of R59527. More than 98% of the sequence thus obtained (SEQ
ID NO: 7) coincided with that of R59527, confirming that a gene
containing the sequence of R59527 is expressed in the adult human
brain.
[0146] (3) Isolation of Rat Semaphorin Y Gene
[0147] Using the 170 bp fragment cloned in (2), which corresponds
to part of R59527, as a probe, the inventors cloned a full-length
cDNA containing the sequence of the probe and determined the
structure. Since preparation of rat cDNA library is easier than
that of human cDNA library, the rat gene was firstly cloned. A cDNA
library was prepared by conventional methods described in the
above-mentioned laboratory manuals, using mRNAs prepared from rat
brain and muscle by conventional procedures with Lambda Zap II
(.lambda.ZapII) cDNA Library Preparation Kit (Stratagene). About
150 thousand plaques were then generated on agar plates using the
cDNA library, and the plaques were transferred onto nylon membranes
(Nippon Pall). After denaturing and neutralizing the DNAs, they
were fixed with ultraviolet rays of 0.6 J/cm.sup.2, and used in
hybridization. The hybridization was conducted by placing the nylon
membrane and the 170 bp DNA fragment labeled with .sup.32P
(prepared using Megaprime DNA Labeling System (Amersham)) as a
probe in a hybridization buffer (45% (v/v) formamide, 5.times.SSPE
(1.times.SSPE consists of 0.15 M sodium chloride, 10 mM sodium
dihydrogenphosphate, and 1 mM ethylenediaminetetraacetic acid
disodium salt, adjusted to pH 7.0), 2.times.Denhardt's solution
(Wako Pure Chemical Industries), 0.5% (w/v) sodium dodecyl sulfate
(SDS), 20 .mu.g/ml salmon sperm DNA (Wako Pure Chemical
Industries)) and allowing them to stand at 42.degree. C. for 48
hours. After the reaction, the nylon membrane was washed 2-3 times
in 2.times.SSPE, 0.5% (w/v) SDS at room temperature for 10 min, and
then 2-3 times in 2.times.SSPE, 0.5% (w/v) SDS at 42.degree. C. for
10 min. The filters thus prepared were analyzed using BAS 2000
Bio-Imaging Analyzer (Fuji Film), and 6 positive signals were
obtained. Plaques corresponding to the positive signals were
excised from the agar plates, placed in 500 .mu.l of SM buffer (100
mM sodium chloride, 15 mM magnesium sulfate, 50 mM Tris (pH 7.5),
0.01% gelatin) supplemented with 20 .mu.l of chloroform, and left
stand overnight at 4.degree. C. to elute the phages. The
recombinant lambda phages thus obtained were subjected to a
secondary screening according to the procedures as described above,
and single plaques were isolated. The phages thus obtained were
treated in the following manner for in vivo excision of a phagemid
containing the cDNA insert, according to the protocols supplied by
Stratagene. Agar gels containing the 4 single plaques obtained in
the secondary screening were each placed in 500 .mu.l of SM buffer,
supplemented with 20 .mu.l of chloroform, and then allowed to stand
overnight at 4.degree. C. Two hundred fifty .mu.l of the phage
solution obtained, 200 .mu.l of E. coli XL-1 Blue MRF' suspended in
10 mM magnesium chloride at OD.sub.600=1.0, and 1 .mu.l of ExAssist
helper phage (>1.times.10.sup.6 pfu/ml) were mixed, and
incubated at 37.degree. C. for 15 min. Then, 3 ml of LB medium
(prepared by mixing 0.5% (w/v) sodium chloride, 1% (w/v)
Bactotrypton (Difco), and 0.5% (w/v) yeast extract (Difco) and the
mixture was adjusting to pH 7.0 using 5 M sodium hydroxide) was
added, and the mixture was shaken at 37.degree. C. for 2-3 hours.
The cells were removed by centrifuging at 2000.times.g for 15 min,
and the supernatant was heat-treated at 70.degree. C. for 15 min.
The supernatant was then centrifuged again at 2000.times.g for 15
min, and recovered as a stock solution of a phagemid containing the
cDNA insert. An aliquot (10-100 .mu.l) of the phagemid stock
solution was mixed with 200 .mu.l of E. coli SOLR (OD.sub.600=1.0),
incubated at 37.degree. C. for 15 min. Then, 10-50 .mu.l of the
mixture was plated onto an ampicillin plate, and incubated
overnight at 37.degree. C. to obtain E. coli strain which contained
the phagemid corresponding to the above positive plaque.
[0148] (4) DNA Sequencing
[0149] The base sequence of the cDNA clone thus obtained was
analyzed on Perkin-Elmer Model 377 DNA Sequencer to determine the
complete base sequence. The reaction was carried out using PRISM
Dye termination kit (Perkin-Elmer). The DNA base sequence thus
determined (3195 bases), the putative open reading frame (2787
bases), and the amino acid sequence (929 amino acids) are shown in
SEQ ID NOs: 1, 2, and 3, respectively.
[0150] The protein contained a so-called semaphorin domain at
positions 46 through 570 in the amino acid sequence, definitely
confirming that the protein belongs to the Semaphorin family. The
protein encoded by the gene was thus designated Semaphorin Y. In
addition, since the base sequence of positions 1574 through 1811 in
the Semaphorin Y gene shown in SEQ ID NO: 1 had 89% identity with
the whole sequence of R59527 consisting of 238 bp, it was confirmed
that R59527 is a partial sequence of human Semaphorin Y gene.
Example 2
Distribution of Rat Semaphorin Y Expression Determined by Northern
Analysis
[0151] In order to determine the distribution of Semaphorin Y gene
expression among rat tissues, RNAs were prepared from various
tissues and used in Northern analysis. RNAs were prepared as
follows using various rat tissues according to AGPC method (Takashi
Tuji and Toshikazu Nakamura, Jikken-Igaku, vol. 9, 1991, pp.
1937-1940; M. F. Ausubel et al. ed., "Current Protocols in
Molecular Biology", 1989, pp. 4.2.4-4.2.8, Greene Pub. Associates
& Wiley-Interscience). Briefly, 10 ml of a denaturing solution
(4M guanidine thiocyanate, 25 mM sodium citrate (pH 7.0), 0.5%
sarkosyl, 0.1 M 2-mercaptoethanol) was added to each 1 g of excised
tissues, and quickly homogenized using a Polytron homogenizer. To
the homogenate, 0.1 volume of 2 M sodium acetate (pH 4.0), 1 volume
of water-saturated phenol, and 0.2 volumes of chloroform-isoamyl
alcohol (49:1) were added, and the mixture was vigorously stirred.
After centrifugation, the aqueous layer was isolated, an equal
volume of isopropyl alcohol was added thereto, and the mixture was
allowed to stand at -20.degree. C. for 1 hour. The precipitate was
recovered by centrifugation, and dissolved again in 2-3 ml of the
denaturing solution per 1 g tissue. An equal volume of isopropyl
alcohol was added, and the mixture was allowed to stand at
-20.degree. C. for 1 hour, and then RNA was centrifuged. The
precipitate was washed with 75% ethyl alcohol, dried briefly, and
then dissolved in an appropriate amount of water.
[0152] Subsequently, electrophoresis and Northern blotting of RNAs
were performed by conventional methods described below. RNAs
prepared from various tissues were firstly electrophoresed on 1%
agarose gel containing formaldehyde. The gel was shaken in 50 mM
NaOH for 20 min, and then in 10.times.SSPE for 40 min. The RNAs
were then blotted onto a nylon membrane (Biodyne B, Nippon Pall) by
means of capillary transfer, and fixed using a UV cross-linker
(Stratagene) (0.6J/cm.sup.2) for use in hybridization. A probe was
prepared as follows. Firstly, PCR was carried out using two
primers, 5' TGTGTAAACGTGACATGG 3' (SEQ ID NO: 10) and 5'
TGCTAGTCAGAGTGAGGA 3' (SEQ ID NO: 11), with rat Semaphorin Y cDNA
obtained in Example 1 as template to amplify a fragment of 477 bp.
This fragment was cloned into pCR II in the same manner as
described above, and the base sequence was determined to confirm
that it was a fragment of rat Semaphorin Y gene. Using this plasmid
DNA as template, PCR was carried out in conventional manner with
the above primers to amplify the aimed fragment of 545 bp. The
amplified DNA separated and purified using agarose gel was labeled
with .sup.32P using Megaprime DNA Labeling System (Amersham) as
described in Example 1, and used as a probe. Hybridization was
carried out by placing the nylon membrane and the probe DNA in the
same hybridization buffer as described above in (2) and allowing
them to stand at 42.degree. C. for 48 hours. After the reaction,
the nylon membrane was washed 2-3 times in 2.times.SSPE, 0.5% (w/v)
SDS for 10 min at 42.degree. C., and then 2-3 times in
2.times.SSPE, 0.5% SDS (w/v) at 55.degree. C. for 10 min.
Radioactivity on the membrane was then analyzed using BAS 2000
Bio-Imaging Analyzer. As shown in FIGS. 1 and 2, the result
demonstrated that mRNA for Semaphorin Y was widely expressed in the
adult CNS, whereas the expression was not detected in peripheral
tissues with the only exception of muscle, exhibiting the
characteristic features expected with Semaphorin gene as CNS-neuron
regeneration inhibitor.
Example 3
Sequence Determination of Human Semaphorin Y
[0153] Since R59527 has proved to be part of human Semaphorin Y
gene as described above, an EST clone containing the sequence of
R59527 (#41581) was obtained from Genome Systems Inc. (US), and the
complete base sequence was determined by the method described
above. The determined base sequence had a high homology to the
entire base sequence for rat Semaphorin Y shown in SEQ ID NO: 1
with 74% of the bases being the same. In addition, the 5' region of
the base sequence contains a stretch, presumably part of the open
reading frame, which could be continuously translated into 427
amino acids. This amino acid sequence had an identity of 82% with
that of the region from position 504 to position 929 of rat
Semaphorin Y gene shown in SEQ ID NO: 3, indicating that the
sequence was certainly part of human Semaphorin Y. However, the
sequence corresponding to the N-terminal of human Semaphorin Y
could not be determined from this clone #41581. In order to
determine the base sequence for human Semaphorin Y in full length,
human hippocampus and forebrain cDNA libraries purchased from
Stratagene were screened as described above using various rat
Semaphorin Y cDNA fragments as probes to obtain a clone #10. The
base sequence of the clone #10 determined by the same procedures as
described above overlapped with the above clone #41581 by about 200
bases, and further contained in its 5' region a cDNA sequence
consisting of more than 1700 bases. The complete base sequence
(3432 bases) constructed from #41581 and #10, the open reading
frame (2790 bases), and the amino acid sequence (930 amino acids)
for human Semaphorin Y are shown in SEQ ID NOs: 4, 5, and 6,
respectively. Human Semaphorin Y was 87% identical at the amino
acid level to rat Semaphorin Y.
[0154] E. coli strain SOLR (hSY10), a transformant obtained by
introducing the plasmid hSY10, which incorporates the insert of the
above clone #10 (the region corresponding to cDNA for human
Semaphorin Y) in a vector pBluescript, into E. coli strain SOLR,
has been deposited at The National Institute of Bioscience and
Human Technology (1-1-3 Higashi, Tsukuba, Ibaraki, Japan) under
Deposit No. FERM BP-6021 on Jul. 11, 1997.
[0155] E. coli strain DH10B (N041581), a transformant obtained by
introducing the plasmid N041581, which incorporates the insert of
the above clone #41581 (the region corresponding to cDNA for human
Semaphorin Y) in a vector Lafmid BA, into E. coli strain DH10B, has
been deposited at The National Institute of Bioscience and Human
Technology (1-1-3 Higashi, Tsukuba, Ibaraki, Japan) under Deposit
No. FERM BP-6022 on Jul. 11, 1997.
Example 4
Distribution of Human Semaphorin Y Expression Determined by
Northern Analysis
[0156] Northern analysis was performed as described in Example 2
with human mRNA blotting membrane (Clontech) using a rat Semaphorin
Y cDNA fragment consisting of 479 bp from position 832 to position
1310 in SEQ ID NO: 1 obtained by PCR as a probe to determine the
distribution of Semaphorin Y mRNA expression among various regions
in human adult CNS tissues. As shown in FIG. 3, human Semaphorin Y
mRNA was widely expressed in various regions of the adult CNS
tissues, and a particularly high expression was observed in the
cerebellum.
[0157] As stated above, Semaphorin Y is widely expressed in human
CNS tissues as is the case with its rat homologue, indicating that
Semaphorin Y may be responsible for functions common to rodents and
primates.
Example 5
Expression of Semaphorin Y in Animal Cells
[0158] A fragment encoding Myc tag having the sequence
Asp-Ile-Gly-Gly-Glu-Gln-Lys-Lue-Ile-Ser-Glu-Glu-Asp-Leu was
inserted just before the stop codon of rat Semaphorin Y gene, and
the recombinant gene was introduced into an expression plasmid
pUCSR.alpha.. The expression plasmid was, transfected into COS 7
cells according to DEAE-dextran method ("Current Protocols in
Molecular Biology" edited by F. M. Ausubel, John Wiley & Sons,
1987), and the cells were harvested with a cell scraper after 48
hours. Harvested cells were homogenized in the presence of Solution
A containing protease inhibitors (Hank's physiological saline
containing 10 mM HEPES pH 7.4, 1 mM EDTA, 50 .mu.M leupeptin, 2
.mu.M pepstatin, 0.5 mM PMSF, and 7.8 mTIU/ml aprotinin) and the
homogenate was separated into precipitate and supernatant by
high-speed centrifugation at 12,000 g for 10 min. The precipitate
from the high-speed centrifugation which contained the membrane
fraction was washed twice with Solution A, suspended in 2 volumes
of 2.25 M sucrose/PBS, and overlaid onto 2.25 M sucrose/PBS. After
0.8 M sucrose/PBS was further overlaid onto the top, it was
centrifuged at 12,000 g for 20 min. The membrane fraction was
recovered from the lower interface, further washed twice, and
stored at -80.degree. C. until use.
[0159] The membrane fraction obtained was subjected to SDS-PAGE
(10%-20% gradient gel), and then to Western blotting in
conventional manner to confirm the production of Semaphorin Y of
the present invention. During this procedure, an anti-Myc antibody
9E10 (Calbiochem) and an alkaline phosphatase-labeled anti-mouse
IgG antibody (Biosource) were used as the primary and secondary
antibodies, respectively. The result of Western blotting showed a
specific band at the position corresponding to about 130 kDa as
shown in FIG. 4, confirming that Myc-tagged rat Semaphorin Y
protein was expressed in COS cells and existed in the membrane.
Example 6
Activity Measurement of Semaphorin Y
[0160] The membrane fraction obtained in Example 5 and another
membrane fraction prepared in the same manner from COS 7 cells
untransfected with Semaphorin Y are each added to culture medium
for neurons such as CNS-neurons or dorsal root ganglion cells, and
the growth-corn collapse activities are compared by the method
described in M. Igarashi et. al., Science, 259, 77-79 (1993). The
result demonstrates that the membrane fraction from COS 7 cells
transfected with the expression plasmid for Semaphorin Y has a
significantly high growth-corn collapse activity.
Reference Example 1
Identification of the Site Essential to the Semaphorin Activity
using Semaphorin III
[0161] PCR was conducted on the basis of the sequence information
on Semaphorin III described in Neuron, 14, 941-948 (1995), and the
structural gene of Semaphorin III was incorporated into an
expression plasmid pUCSR.alpha.. The expression plasmid was then
introduced into COS 7 cells by DEAE-dextran method. After 2 days,
the Semaphorin III activity contained in the culture supernatant
was determined by a method similar to that described in Cell, 75,
217-227 (1993), using the growth-corn collapse activity on chicken
dorsal root ganglion cells as an indicator. As a result, one clone
which did not exhibit any activity was found. The base sequencing
of the clone revealed that aspartic acid residue at position 198
was substituted by glycine. When compared with other known animal
Semaphorins, the regions before and after the position 198 were not
markedly conserved, although the position corresponding to aspartic
acid was highly conserved among Semaphorins with a few exceptions
in which glutamic acid was located at that position. This suggested
that the aspartic acid residue is essential to expression of the
activity. The gene was then subjected to a site-directed
mutagenesis by a conventional method to replace the glycine residue
with aspartic acid. Since this mutagenesis restored the strong
collapse activity, it was confirmed that all of the regions in the
expression plasmid normally function except for that position. In
conclusion, the aspartic acid at position 198 of Semaphorin III
appears essential to expression of the Semaphorin function. The
amino acid residues corresponding to the aspartic acid are aspartic
acid at position 197 in the amino acid sequence of rat Semaphorin Y
shown in SEQ ID NO: 3, and aspartic acid at position 198 in the
amino acid sequence of human Semaphorin Y shown in SEQ ID NO:
6.
Reference Example 2
Tissue-specific Gene Expression of Semaphorin III Determined by
Northern Analysis
[0162] To determine the distribution of Semaphorin III gene
expression among mouse tissues, RNAs were prepared from various
adult mouse tissues, and subjected to Northern analysis. The
procedures for preparation, blotting, and hybridization of RNA were
the same as those described in Example 2., As a probe, the 560 bp
MspI fragment of mouse Semaphorin III DNA described in Reference
example 1 was used. As a result, it was demonstrated as shown in
FIG. 5 that the expression of Semaphorin III in the adult is
extremely high in the lung, while it is rather low in the CNS.
EFFECTS OF THE INVENTION
[0163] The present invention provides Semaphorin Y inhibiting
neurite outgrowth, and a gene therefor, as well as other
Semaphorins hybridizing to said Semaphorin Y gene, modified
proteins or partial peptides of said Semaphorin Y, antibodies
against said Semaphorin Y, antisense nucleotides against said
Semaphorin Y gene, and the use of such substances as pharmaceutical
or diagnostic agents or laboratory reagents. The present invention
further provides a method of screening for Semaphorin Y antagonists
employing said Semaphorin Y, Semaphorin Y antagonists obtained by
said screening method, pharmaceutical agents comprising such
antagonists, and transgenic animals involving said Semaphorin Y.
Sequence CWU 1
1
13 1 3195 DNA Rattus norvegicus misc_feature (1)..(3195)
Strandedness Double-stranded 1 gcggccgcgt cgacggggta ctccctgggg
cttgagctgc cccgcacagg atgccccgtg 60 ccccccactc catgcccttg
ctgctgctgt tgctgctgtc actcccccaa gcccagactg 120 cctttcccca
ggaccccatc cctctgttga cctctgacct acaaggtacc tctccgtcat 180
cctggttccg gggcctggag gacgatgctg tggctgcgga acttgggctg gactttcaga
240 gattcctgac cttgaaccgg accttgcttg tggctgcccg ggatcacgtt
ttctccttcg 300 atcttcaagc ccaagaagaa ggggaggggc tggtgcccaa
caagtttctg acatggcgga 360 gccaagacat ggagaattgt gctgtccggg
gaaagctgac ggacgaatgc tacaactaca 420 tccgtgttct tgttccctgg
gactcgcaga cactccttgc ctgtggaaca aattccttca 480 gccctgtgtg
tcgcagctat gggataacat ctctgcaaca ggagggtgag gagctgagtg 540
ggcaagctcg atgccccttt gatgccaccc agtccactgt ggccatctct gcagagggta
600 gtttgtactc agccacagca gcagatttcc aggccagtga tgctgtggtt
tacagaagcc 660 ttggacctca gcccccactc cgttctgcaa agtatgactc
caagtggctt cgagagccac 720 actttgtcta tgctttggag catggagacc
atgtctactt ctttcttccg gagaagtctc 780 tgtggaggac gcccggcctg
gggagggtgc agttttcccg ggtggcccgg gtgtgtaaac 840 gtgacatggg
tggctcacca cgggccttgg atcgccactg gacatccttc cttaagctga 900
ggctcaactg ctccgtccct ggggactcta ccttctactt tgatgtctta cagtccttaa
960 ctgggcctgt gaacctgcat gggcgctctg ccctctttgg ggtcttcact
actcagacca 1020 atagcattcc tgggtctgca gtctgcgcct tctacctaga
tgacattgaa cgtggctttg 1080 agggcaagtt caaggagcag aggagtctgg
atggggcctg gactcctgtg tctgaggaca 1140 aagtcccctc acccaggcca
gggtcctgtg caggtgtggg tgcagctgcc ttattctcct 1200 cctctcaaga
cctgcctgac gatgtcctgc tcttcatcaa ggcacaccca ctgctggatc 1260
ccgctgtgcc acctgccacc catcaacctc tcctcactct gactagcagg gctctactga
1320 cccaggtagc tgtggatggt atggctggcc cccacagaaa tactacagtc
ctgtttcttg 1380 gctccaatga tgggacagtg ctgaaggtgc tacctccagg
gggacagtct ctgggacccg 1440 agcctatcat attggaagag attgatgcct
acagccatgc ccggtgcagt gggaagcggt 1500 caccccgagc tgctcgacgg
atcatagggc tggagctgga cactgagggt cacaggcttt 1560 ttgtggcctt
tcctggatgc atcgtctacc tctctctcag ccgctgtgcc cggcatggag 1620
catgtcagag gagctgcctg gcttctctgg acccatactg tggatggcat cggttccgag
1680 gctgtgtgaa tatcagggga cctggaggga ctgatgtgga tctgactggg
aaccaggaat 1740 ccatggagca tggtgactgc caagatggag cgactgggag
tcagtctggc cctggagatt 1800 ctgcctatgg cgtgcgcagg gacctttccc
cagcctcagc ctcccgatcc atccccatcc 1860 cactcctcct ggcctgtgtg
gcggcggcct tcgctttggg cgcctcagtc tccggcctct 1920 tggtgtcctg
tgcttgtcgt cgcgcgaacc gccgtcggag caaggacatc gagaccccgg 1980
ggctgccgcg ccccctctcc cttcgcagtc tggcgaggct gcacggtggc ggtcctgagc
2040 ccccgcctcc gcccaaggat ggtgatgcag cgcaaacgcc ccagctctac
actaccttcc 2100 tgcctccgcc cgagggcgga tccccaccgg agctggcctg
cctgcccacc ccggagacca 2160 cgcccgagct gccggtgaag cacctccgtg
cctccggggg tccctgggag tggaaccaga 2220 acgggaacaa cgcttcggag
ggcccaggcc gcccacgggg ctgcagcgcg gcgggcgggc 2280 ccgccccgcg
cgtgctggtg aggccaccgc cccctggctg ccccgggcag gaggtggagg 2340
tgaccacgct ggaggaactg ctgcgctacc tgcacggccc gcagccgccc aggaagggca
2400 gcgaacctct cgcctccgcc ccgttcacct cccggccgcc tgcctcggag
cccggcgccg 2460 ccttgttcgt ggactccagc ccgatgcctc gtgattgcgt
gccgccgctg aggctcgacg 2520 taccgcccga cggcaagcgc gcggccccga
gcgggcggcc tgctctctcg gccccggctc 2580 cacgcctggg cgtcagcggc
agccgaagat tgcccttccc cacgcaccgg gcgcccccgg 2640 gcctgctcac
ccgagtcccc tcgggaggcc cgtccaggta ctccgggggg cccgggaggc 2700
acctcctgta cctgggccgg cccgacggcc accgcggccg ctccctgaag agggtggacg
2760 tgaagtctcc actgtcgccc aaaccgcccc tcgccacacc gccgcagccc
gccccgcacg 2820 gcagccattt taacttctga cagaagctgc tagcgcccgt
cgaggcgctg gaggcctagg 2880 cctgcggagg ccgctggcct tcccggactc
caagagtctc ccggggtccc ctctcgcctc 2940 ggtttattta ttgactgtct
ttccccctgt cctttggcga ggagctcgcc gctcggagcg 3000 ccagcatttc
aggggacctg gccgactccc actccccgct cccttccagc cacgctgcct 3060
taactcgtcg ctccggactc ccgcggactg ggccccgggc gggccggccg gggctggagc
3120 cgcgcgctgt gtacagagtc ctccggcctc ctggggccgg gacgtgcctc
ctcctactgt 3180 gtaggagccc ccacc 3195 2 2787 DNA Rattus norvegicus
misc_feature (1)..(2787) Strandedness Double-stranded 2 atg ccc cgt
gcc ccc cac tcc atg ccc ttg ctg ctg ctg ttg ctg ctg 48 Met Pro Arg
Ala Pro His Ser Met Pro Leu Leu Leu Leu Leu Leu Leu 1 5 10 15 tca
ctc ccc caa gcc cag act gcc ttt ccc cag gac ccc atc cct ctg 96 Ser
Leu Pro Gln Ala Gln Thr Ala Phe Pro Gln Asp Pro Ile Pro Leu 20 25
30 ttg acc tct gac cta caa ggt acc tct ccg tca tcc tgg ttc cgg ggc
144 Leu Thr Ser Asp Leu Gln Gly Thr Ser Pro Ser Ser Trp Phe Arg Gly
35 40 45 ctg gag gac gat gct gtg gct gcg gaa ctt ggg ctg gac ttt
cag aga 192 Leu Glu Asp Asp Ala Val Ala Ala Glu Leu Gly Leu Asp Phe
Gln Arg 50 55 60 ttc ctg acc ttg aac cgg acc ttg ctt gtg gct gcc
cgg gat cac gtt 240 Phe Leu Thr Leu Asn Arg Thr Leu Leu Val Ala Ala
Arg Asp His Val 65 70 75 80 ttc tcc ttc gat ctt caa gcc caa gaa gaa
ggg gag ggg ctg gtg ccc 288 Phe Ser Phe Asp Leu Gln Ala Gln Glu Glu
Gly Glu Gly Leu Val Pro 85 90 95 aac aag ttt ctg aca tgg cgg agc
caa gac atg gag aat tgt gct gtc 336 Asn Lys Phe Leu Thr Trp Arg Ser
Gln Asp Met Glu Asn Cys Ala Val 100 105 110 cgg gga aag ctg acg gac
gaa tgc tac aac tac atc cgt gtt ctt gtt 384 Arg Gly Lys Leu Thr Asp
Glu Cys Tyr Asn Tyr Ile Arg Val Leu Val 115 120 125 ccc tgg gac tcg
cag aca ctc ctt gcc tgt gga aca aat tcc ttc agc 432 Pro Trp Asp Ser
Gln Thr Leu Leu Ala Cys Gly Thr Asn Ser Phe Ser 130 135 140 cct gtg
tgt cgc agc tat ggg ata aca tct ctg caa cag gag ggt gag 480 Pro Val
Cys Arg Ser Tyr Gly Ile Thr Ser Leu Gln Gln Glu Gly Glu 145 150 155
160 gag ctg agt ggg caa gct cga tgc ccc ttt gat gcc acc cag tcc act
528 Glu Leu Ser Gly Gln Ala Arg Cys Pro Phe Asp Ala Thr Gln Ser Thr
165 170 175 gtg gcc atc tct gca gag ggt agt ttg tac tca gcc aca gca
gca gat 576 Val Ala Ile Ser Ala Glu Gly Ser Leu Tyr Ser Ala Thr Ala
Ala Asp 180 185 190 ttc cag gcc agt gat gct gtg gtt tac aga agc ctt
gga cct cag ccc 624 Phe Gln Ala Ser Asp Ala Val Val Tyr Arg Ser Leu
Gly Pro Gln Pro 195 200 205 cca ctc cgt tct gca aag tat gac tcc aag
tgg ctt cga gag cca cac 672 Pro Leu Arg Ser Ala Lys Tyr Asp Ser Lys
Trp Leu Arg Glu Pro His 210 215 220 ttt gtc tat gct ttg gag cat gga
gac cat gtc tac ttc ttt ctt ccg 720 Phe Val Tyr Ala Leu Glu His Gly
Asp His Val Tyr Phe Phe Leu Pro 225 230 235 240 gag aag tct ctg tgg
agg acg ccc ggc ctg ggg agg gtg cag ttt tcc 768 Glu Lys Ser Leu Trp
Arg Thr Pro Gly Leu Gly Arg Val Gln Phe Ser 245 250 255 cgg gtg gcc
cgg gtg tgt aaa cgt gac atg ggt ggc tca cca cgg gcc 816 Arg Val Ala
Arg Val Cys Lys Arg Asp Met Gly Gly Ser Pro Arg Ala 260 265 270 ttg
gat cgc cac tgg aca tcc ttc ctt aag ctg agg ctc aac tgc tcc 864 Leu
Asp Arg His Trp Thr Ser Phe Leu Lys Leu Arg Leu Asn Cys Ser 275 280
285 gtc cct ggg gac tct acc ttc tac ttt gat gtc tta cag tcc tta act
912 Val Pro Gly Asp Ser Thr Phe Tyr Phe Asp Val Leu Gln Ser Leu Thr
290 295 300 ggg cct gtg aac ctg cat ggg cgc tct gcc ctc ttt ggg gtc
ttc act 960 Gly Pro Val Asn Leu His Gly Arg Ser Ala Leu Phe Gly Val
Phe Thr 305 310 315 320 act cag acc aat agc att cct ggg tct gca gtc
tgc gcc ttc tac cta 1008 Thr Gln Thr Asn Ser Ile Pro Gly Ser Ala
Val Cys Ala Phe Tyr Leu 325 330 335 gat gac att gaa cgt ggc ttt gag
ggc aag ttc aag gag cag agg agt 1056 Asp Asp Ile Glu Arg Gly Phe
Glu Gly Lys Phe Lys Glu Gln Arg Ser 340 345 350 ctg gat ggg gcc tgg
act cct gtg tct gag gac aaa gtc ccc tca ccc 1104 Leu Asp Gly Ala
Trp Thr Pro Val Ser Glu Asp Lys Val Pro Ser Pro 355 360 365 agg cca
ggg tcc tgt gca ggt gtg ggt gca gct gcc tta ttc tcc tcc 1152 Arg
Pro Gly Ser Cys Ala Gly Val Gly Ala Ala Ala Leu Phe Ser Ser 370 375
380 tct caa gac ctg cct gac gat gtc ctg ctc ttc atc aag gca cac cca
1200 Ser Gln Asp Leu Pro Asp Asp Val Leu Leu Phe Ile Lys Ala His
Pro 385 390 395 400 ctg ctg gat ccc gct gtg cca cct gcc acc cat caa
cct ctc ctc act 1248 Leu Leu Asp Pro Ala Val Pro Pro Ala Thr His
Gln Pro Leu Leu Thr 405 410 415 ctg act agc agg gct cta ctg acc cag
gta gct gtg gat ggt atg gct 1296 Leu Thr Ser Arg Ala Leu Leu Thr
Gln Val Ala Val Asp Gly Met Ala 420 425 430 ggc ccc cac aga aat act
aca gtc ctg ttt ctt ggc tcc aat gat ggg 1344 Gly Pro His Arg Asn
Thr Thr Val Leu Phe Leu Gly Ser Asn Asp Gly 435 440 445 aca gtg ctg
aag gtg cta cct cca ggg gga cag tct ctg gga ccc gag 1392 Thr Val
Leu Lys Val Leu Pro Pro Gly Gly Gln Ser Leu Gly Pro Glu 450 455 460
cct atc ata ttg gaa gag att gat gcc tac agc cat gcc cgg tgc agt
1440 Pro Ile Ile Leu Glu Glu Ile Asp Ala Tyr Ser His Ala Arg Cys
Ser 465 470 475 480 ggg aag cgg tca ccc cga gct gct cga cgg atc ata
ggg ctg gag ctg 1488 Gly Lys Arg Ser Pro Arg Ala Ala Arg Arg Ile
Ile Gly Leu Glu Leu 485 490 495 gac act gag ggt cac agg ctt ttt gtg
gcc ttt cct gga tgc atc gtc 1536 Asp Thr Glu Gly His Arg Leu Phe
Val Ala Phe Pro Gly Cys Ile Val 500 505 510 tac ctc tct ctc agc cgc
tgt gcc cgg cat gga gca tgt cag agg agc 1584 Tyr Leu Ser Leu Ser
Arg Cys Ala Arg His Gly Ala Cys Gln Arg Ser 515 520 525 tgc ctg gct
tct ctg gac cca tac tgt gga tgg cat cgg ttc cga ggc 1632 Cys Leu
Ala Ser Leu Asp Pro Tyr Cys Gly Trp His Arg Phe Arg Gly 530 535 540
tgt gtg aat atc agg gga cct gga ggg act gat gtg gat ctg act ggg
1680 Cys Val Asn Ile Arg Gly Pro Gly Gly Thr Asp Val Asp Leu Thr
Gly 545 550 555 560 aac cag gaa tcc atg gag cat ggt gac tgc caa gat
gga gcg act ggg 1728 Asn Gln Glu Ser Met Glu His Gly Asp Cys Gln
Asp Gly Ala Thr Gly 565 570 575 agt cag tct ggc cct gga gat tct gcc
tat ggc gtg cgc agg gac ctt 1776 Ser Gln Ser Gly Pro Gly Asp Ser
Ala Tyr Gly Val Arg Arg Asp Leu 580 585 590 tcc cca gcc tca gcc tcc
cga tcc atc ccc atc cca ctc ctc ctg gcc 1824 Ser Pro Ala Ser Ala
Ser Arg Ser Ile Pro Ile Pro Leu Leu Leu Ala 595 600 605 tgt gtg gcg
gcg gcc ttc gct ttg ggc gcc tca gtc tcc ggc ctc ttg 1872 Cys Val
Ala Ala Ala Phe Ala Leu Gly Ala Ser Val Ser Gly Leu Leu 610 615 620
gtg tcc tgt gct tgt cgt cgc gcg aac cgc cgt cgg agc aag gac atc
1920 Val Ser Cys Ala Cys Arg Arg Ala Asn Arg Arg Arg Ser Lys Asp
Ile 625 630 635 640 gag acc ccg ggg ctg ccg cgc ccc ctc tcc ctt cgc
agt ctg gcg agg 1968 Glu Thr Pro Gly Leu Pro Arg Pro Leu Ser Leu
Arg Ser Leu Ala Arg 645 650 655 ctg cac ggt ggc ggt cct gag ccc ccg
cct ccg ccc aag gat ggt gat 2016 Leu His Gly Gly Gly Pro Glu Pro
Pro Pro Pro Pro Lys Asp Gly Asp 660 665 670 gca gcg caa acg ccc cag
ctc tac act acc ttc ctg cct ccg ccc gag 2064 Ala Ala Gln Thr Pro
Gln Leu Tyr Thr Thr Phe Leu Pro Pro Pro Glu 675 680 685 ggc gga tcc
cca ccg gag ctg gcc tgc ctg ccc acc ccg gag acc acg 2112 Gly Gly
Ser Pro Pro Glu Leu Ala Cys Leu Pro Thr Pro Glu Thr Thr 690 695 700
ccc gag ctg ccg gtg aag cac ctc cgt gcc tcc ggg ggt ccc tgg gag
2160 Pro Glu Leu Pro Val Lys His Leu Arg Ala Ser Gly Gly Pro Trp
Glu 705 710 715 720 tgg aac cag aac ggg aac aac gct tcg gag ggc cca
ggc cgc cca cgg 2208 Trp Asn Gln Asn Gly Asn Asn Ala Ser Glu Gly
Pro Gly Arg Pro Arg 725 730 735 ggc tgc agc gcg gcg ggc ggg ccc gcc
ccg cgc gtg ctg gtg agg cca 2256 Gly Cys Ser Ala Ala Gly Gly Pro
Ala Pro Arg Val Leu Val Arg Pro 740 745 750 ccg ccc cct ggc tgc ccc
ggg cag gag gtg gag gtg acc acg ctg gag 2304 Pro Pro Pro Gly Cys
Pro Gly Gln Glu Val Glu Val Thr Thr Leu Glu 755 760 765 gaa ctg ctg
cgc tac ctg cac ggc ccg cag ccg ccc agg aag ggc agc 2352 Glu Leu
Leu Arg Tyr Leu His Gly Pro Gln Pro Pro Arg Lys Gly Ser 770 775 780
gaa cct ctc gcc tcc gcc ccg ttc acc tcc cgg ccg cct gcc tcg gag
2400 Glu Pro Leu Ala Ser Ala Pro Phe Thr Ser Arg Pro Pro Ala Ser
Glu 785 790 795 800 ccc ggc gcc gcc ttg ttc gtg gac tcc agc ccg atg
cct cgt gat tgc 2448 Pro Gly Ala Ala Leu Phe Val Asp Ser Ser Pro
Met Pro Arg Asp Cys 805 810 815 gtg ccg ccg ctg agg ctc gac gta ccg
ccc gac ggc aag cgc gcg gcc 2496 Val Pro Pro Leu Arg Leu Asp Val
Pro Pro Asp Gly Lys Arg Ala Ala 820 825 830 ccg agc ggg cgg cct gct
ctc tcg gcc ccg gct cca cgc ctg ggc gtc 2544 Pro Ser Gly Arg Pro
Ala Leu Ser Ala Pro Ala Pro Arg Leu Gly Val 835 840 845 agc ggc agc
cga aga ttg ccc ttc ccc acg cac cgg gcg ccc ccg ggc 2592 Ser Gly
Ser Arg Arg Leu Pro Phe Pro Thr His Arg Ala Pro Pro Gly 850 855 860
ctg ctc acc cga gtc ccc tcg gga ggc ccg tcc agg tac tcc ggg ggg
2640 Leu Leu Thr Arg Val Pro Ser Gly Gly Pro Ser Arg Tyr Ser Gly
Gly 865 870 875 880 ccc ggg agg cac ctc ctg tac ctg ggc cgg ccc gac
ggc cac cgc ggc 2688 Pro Gly Arg His Leu Leu Tyr Leu Gly Arg Pro
Asp Gly His Arg Gly 885 890 895 cgc tcc ctg aag agg gtg gac gtg aag
tct cca ctg tcg ccc aaa ccg 2736 Arg Ser Leu Lys Arg Val Asp Val
Lys Ser Pro Leu Ser Pro Lys Pro 900 905 910 ccc ctc gcc aca ccg ccg
cag ccc gcc ccg cac ggc agc cat ttt aac 2784 Pro Leu Ala Thr Pro
Pro Gln Pro Ala Pro His Gly Ser His Phe Asn 915 920 925 ttc 2787
Phe 3 929 PRT Rattus norvegicus misc_feature ()..() Tissue Type
Brain 3 Met Pro Arg Ala Pro His Ser Met Pro Leu Leu Leu Leu Leu Leu
Leu 1 5 10 15 Ser Leu Pro Gln Ala Gln Thr Ala Phe Pro Gln Asp Pro
Ile Pro Leu 20 25 30 Leu Thr Ser Asp Leu Gln Gly Thr Ser Pro Ser
Ser Trp Phe Arg Gly 35 40 45 Leu Glu Asp Asp Ala Val Ala Ala Glu
Leu Gly Leu Asp Phe Gln Arg 50 55 60 Phe Leu Thr Leu Asn Arg Thr
Leu Leu Val Ala Ala Arg Asp His Val 65 70 75 80 Phe Ser Phe Asp Leu
Gln Ala Gln Glu Glu Gly Glu Gly Leu Val Pro 85 90 95 Asn Lys Phe
Leu Thr Trp Arg Ser Gln Asp Met Glu Asn Cys Ala Val 100 105 110 Arg
Gly Lys Leu Thr Asp Glu Cys Tyr Asn Tyr Ile Arg Val Leu Val 115 120
125 Pro Trp Asp Ser Gln Thr Leu Leu Ala Cys Gly Thr Asn Ser Phe Ser
130 135 140 Pro Val Cys Arg Ser Tyr Gly Ile Thr Ser Leu Gln Gln Glu
Gly Glu 145 150 155 160 Glu Leu Ser Gly Gln Ala Arg Cys Pro Phe Asp
Ala Thr Gln Ser Thr 165 170 175 Val Ala Ile Ser Ala Glu Gly Ser Leu
Tyr Ser Ala Thr Ala Ala Asp 180 185 190 Phe Gln Ala Ser Asp Ala Val
Val Tyr Arg Ser Leu Gly Pro Gln Pro 195 200 205 Pro Leu Arg Ser Ala
Lys Tyr Asp Ser Lys Trp Leu Arg Glu Pro His 210 215 220 Phe Val Tyr
Ala Leu Glu His Gly Asp His Val Tyr Phe Phe Leu Pro 225 230 235 240
Glu Lys Ser Leu Trp Arg Thr Pro Gly Leu Gly Arg Val Gln Phe Ser 245
250 255 Arg Val Ala Arg Val Cys Lys Arg Asp Met Gly Gly Ser Pro Arg
Ala 260 265 270 Leu Asp Arg His Trp Thr Ser Phe Leu Lys Leu Arg Leu
Asn Cys Ser 275 280 285 Val Pro Gly Asp Ser Thr Phe Tyr Phe Asp Val
Leu Gln Ser Leu Thr 290 295 300 Gly Pro Val Asn Leu His Gly Arg Ser
Ala Leu Phe Gly Val Phe Thr 305 310 315 320 Thr Gln Thr Asn Ser Ile
Pro Gly Ser Ala Val Cys Ala Phe Tyr Leu 325 330 335 Asp Asp Ile Glu
Arg Gly Phe Glu Gly Lys Phe Lys Glu Gln Arg Ser 340 345 350 Leu Asp
Gly Ala Trp Thr Pro Val Ser Glu Asp Lys Val Pro Ser Pro 355 360 365
Arg Pro Gly Ser Cys Ala Gly Val Gly Ala Ala Ala Leu Phe Ser Ser 370
375 380 Ser Gln
Asp Leu Pro Asp Asp Val Leu Leu Phe Ile Lys Ala His Pro 385 390 395
400 Leu Leu Asp Pro Ala Val Pro Pro Ala Thr His Gln Pro Leu Leu Thr
405 410 415 Leu Thr Ser Arg Ala Leu Leu Thr Gln Val Ala Val Asp Gly
Met Ala 420 425 430 Gly Pro His Arg Asn Thr Thr Val Leu Phe Leu Gly
Ser Asn Asp Gly 435 440 445 Thr Val Leu Lys Val Leu Pro Pro Gly Gly
Gln Ser Leu Gly Pro Glu 450 455 460 Pro Ile Ile Leu Glu Glu Ile Asp
Ala Tyr Ser His Ala Arg Cys Ser 465 470 475 480 Gly Lys Arg Ser Pro
Arg Ala Ala Arg Arg Ile Ile Gly Leu Glu Leu 485 490 495 Asp Thr Glu
Gly His Arg Leu Phe Val Ala Phe Pro Gly Cys Ile Val 500 505 510 Tyr
Leu Ser Leu Ser Arg Cys Ala Arg His Gly Ala Cys Gln Arg Ser 515 520
525 Cys Leu Ala Ser Leu Asp Pro Tyr Cys Gly Trp His Arg Phe Arg Gly
530 535 540 Cys Val Asn Ile Arg Gly Pro Gly Gly Thr Asp Val Asp Leu
Thr Gly 545 550 555 560 Asn Gln Glu Ser Met Glu His Gly Asp Cys Gln
Asp Gly Ala Thr Gly 565 570 575 Ser Gln Ser Gly Pro Gly Asp Ser Ala
Tyr Gly Val Arg Arg Asp Leu 580 585 590 Ser Pro Ala Ser Ala Ser Arg
Ser Ile Pro Ile Pro Leu Leu Leu Ala 595 600 605 Cys Val Ala Ala Ala
Phe Ala Leu Gly Ala Ser Val Ser Gly Leu Leu 610 615 620 Val Ser Cys
Ala Cys Arg Arg Ala Asn Arg Arg Arg Ser Lys Asp Ile 625 630 635 640
Glu Thr Pro Gly Leu Pro Arg Pro Leu Ser Leu Arg Ser Leu Ala Arg 645
650 655 Leu His Gly Gly Gly Pro Glu Pro Pro Pro Pro Pro Lys Asp Gly
Asp 660 665 670 Ala Ala Gln Thr Pro Gln Leu Tyr Thr Thr Phe Leu Pro
Pro Pro Glu 675 680 685 Gly Gly Ser Pro Pro Glu Leu Ala Cys Leu Pro
Thr Pro Glu Thr Thr 690 695 700 Pro Glu Leu Pro Val Lys His Leu Arg
Ala Ser Gly Gly Pro Trp Glu 705 710 715 720 Trp Asn Gln Asn Gly Asn
Asn Ala Ser Glu Gly Pro Gly Arg Pro Arg 725 730 735 Gly Cys Ser Ala
Ala Gly Gly Pro Ala Pro Arg Val Leu Val Arg Pro 740 745 750 Pro Pro
Pro Gly Cys Pro Gly Gln Glu Val Glu Val Thr Thr Leu Glu 755 760 765
Glu Leu Leu Arg Tyr Leu His Gly Pro Gln Pro Pro Arg Lys Gly Ser 770
775 780 Glu Pro Leu Ala Ser Ala Pro Phe Thr Ser Arg Pro Pro Ala Ser
Glu 785 790 795 800 Pro Gly Ala Ala Leu Phe Val Asp Ser Ser Pro Met
Pro Arg Asp Cys 805 810 815 Val Pro Pro Leu Arg Leu Asp Val Pro Pro
Asp Gly Lys Arg Ala Ala 820 825 830 Pro Ser Gly Arg Pro Ala Leu Ser
Ala Pro Ala Pro Arg Leu Gly Val 835 840 845 Ser Gly Ser Arg Arg Leu
Pro Phe Pro Thr His Arg Ala Pro Pro Gly 850 855 860 Leu Leu Thr Arg
Val Pro Ser Gly Gly Pro Ser Arg Tyr Ser Gly Gly 865 870 875 880 Pro
Gly Arg His Leu Leu Tyr Leu Gly Arg Pro Asp Gly His Arg Gly 885 890
895 Arg Ser Leu Lys Arg Val Asp Val Lys Ser Pro Leu Ser Pro Lys Pro
900 905 910 Pro Leu Ala Thr Pro Pro Gln Pro Ala Pro His Gly Ser His
Phe Asn 915 920 925 Phe 4 3432 DNA Homo sapiens misc_feature
(1)..(3432) Strandedness Double-stranded 4 aaaaccacgg attgcgaact
cagcgcagcg cgtggccgct ggccgcccgc ggcgatctcg 60 atcccgctga
cccgaatcct ggagtcagag gtttcctatc cccctcaagc ccccacagga 120
gtcaccaacc cagggccggc ttatgggtga gggggcaccc cctggggcct gagctgcccc
180 acacaggatg ccccgtgccc cccacttcat gcccttgctg ctactgctgc
tgctgctctc 240 acttccccat actcaggccg cctttcccca ggaccccctc
cctctgttga tctctgacct 300 tcaaggtact tccccattat cctggtttcg
gggcctggag gatgatgctg tggctgcaga 360 acttgggctg gactttcaga
gattcctgac cttgaaccgg accttgctag tggctgcccg 420 ggatcacgtt
ttctccttcg atcttcaagc cgaagaagaa ggggaggggc tggtgcccaa 480
caagtatcta acatggagaa gccaagatgt ggagaactgt gctgtacggg gaaagctgac
540 ggatgagtgc tacaactata ttcgtgttct tgttccctgg gactcccaga
cgctccttgc 600 ctgtggaacg aactcattca gccctgtgtg ccgcagctat
gggataactt cgctgcagca 660 ggagggtgag gaactgagtg ggcaggctcg
atgccccttt gatgccaccc agtccaacgt 720 ggccatcttt gcagagggca
gcctgtactc agccacagct gcggatttcc aggccagtga 780 tgctgtagtt
tacagaagcc ttgggcccca gcccccactc cgctccgcca agtatgactc 840
caagtggctc cgagagccac actttgtcca ggccttggag catggagacc atgtctactt
900 cttcttccgc gaggtctctg tggaggatgc tcggctgggg aaggtgcagt
tctcccgcgt 960 agcccgagta tgtaaacgtg acatgggcgg ctcgcctcgg
gccttggacc gccactggac 1020 atccttcctg aagcttcggc tcaactgctc
tgtccctggg gactctactt tctattttga 1080 tgttttacag gccttgactg
ggcctgtgaa cctgcatggc cgctctgctc tctttggggt 1140 cttcaccacc
cagaccaata gcatccctgg ctctgccgtc tgcgccttct acctggatga 1200
gattgagcgt gggtttgagg gcaagttcaa ggagcagagg agtctggatg gggcctggac
1260 tcctgtgtct gaggacagag ttccctcacc caggccagga tcctgtgcag
gagtaggggg 1320 agctgccttg ttctcctctt cccgagacct ccctgatgat
gtcctgacct tcatcaaggc 1380 tcacccgctg ctggaccccg ctgtaccacc
tgtcacccat cagcctctac tcactctcac 1440 tagcagggcc ctactgaccc
aagtagctgt ggatggcatg gctggtcccc acagtaacat 1500 cacagtcatg
ttccttggct ccaatgatgg gacagtgctg aaggtgctga ccccaggtgg 1560
gcgatccggg ggacctgagc ccatcctcct ggaagagatt gatgcctaca gccctgcccg
1620 gtgcagtggg aagcggacag cccaaacagc acgacggatc atagggctgg
agctggacac 1680 tgagggtcac aggctttttg tggctttttc tggctgtatt
gtctacctcc ctctcagccg 1740 gtgtgcccgg catggggcct gtcagaggag
ctgtttggct tctcaggacc catactgtgg 1800 atggcatagc tccaggggct
gtgtggatat caggggatct ggtgggactg atgtggatca 1860 ggctgggaac
caggaatcca tggagcatgg tgactgccaa gatggagcta ctgggagtca 1920
gtctggccct ggggattctg cttatggcgt gcgccgggac ctgcccccag cctcggcctc
1980 ccgctccgtc cccatcccac tcctcctggc cagtgtggcc gcagcttttg
ccctgggcgc 2040 ctcagtctct ggcctcctgg tctcctgtgc ttgtcgccgc
gcccaccgac gtcggggcaa 2100 ggacatcgag actcccgggc tcccgcgccc
tctctccctc cgcagtttgg cccggctcca 2160 cggtgggggc ccagagcccc
cgccgccctc caaggacggg gacgcggtgc agacgccgca 2220 gctctacacc
accttcctgc cgcctccgga gggcgtgccc ccgccggagc tggcctgcct 2280
gcccaccccc gagtccacgc cggagctgcc ggtcaagcac ctccgcgccg ccggggaccc
2340 ctgggagtgg aaccagaaca ggaacaacgc caaggagggt ccgggccgct
cacggggcgg 2400 gcacgcggcg ggcgggcccg cgccccgcgt gctggtgagg
ccaccgccgc ccggctgtcc 2460 cgggcaggcc gtggaagtca ccaccctgga
ggaactgctg cgctacctgc acggcccgca 2520 gccgcccaga aagggggccg
agccccccgc ccctttaacc tcgcgggcgc tcccgccgga 2580 gcccgccccc
gccctcttgg gcggccccag ccccaggccc cacgagtgcg cctcgccgct 2640
gaggctggac gtgccccccg agggcaggtg cgcctctgcc cccgcccggc ccgcgctctc
2700 cgcccccgct ccccggctgg gcgtcggcgg aggccggagg ttgcctttct
ccggccaccg 2760 ggccccccct gccctgctca ctcgagtccc ctcgggaggt
ccctccaggt actccggggg 2820 tcccgggaag cacctcctgt acctgggccg
gcccgagggc taccggggcc gcgccctgaa 2880 aagggtggac gtcgagaagc
cccagttgtc cctgaagcct cccctcgtcg ggccctcctc 2940 ccgccaggcc
gtcccgaacg gcggccgttt caacttttaa agggagcggt ccacggcctc 3000
cagcgtgggg agcgcccgag tcctctcggt cacgagctgg acgctcttca ggacgtttca
3060 ccgccccctc gccccgcacc tccagccttc ccgactcgca gagtctcccg
aggccccttt 3120 tcgcctcggg tttatttatt gactgtcttt ccccctgtcc
tcgacagaag agtgggaggt 3180 gagaagcccg tctcctcagt gagccagcat
ttcaggggga gctggcggac tcccactccc 3240 cgctcccttc cagccaagct
gccttaactc gcccctcggg gctcccccag agactgtgcc 3300 ccgggcgggc
cgcgcgcgct gtgtccagag tcctcgggcc tcctgggtct gggacgtgcc 3360
tctcctactg tgtaggagcc tccgcttccc aatacagccg tgtctgcaaa aaaaaaaaaa
3420 aaaaaaaaaa aa 3432 5 2790 DNA Homo sapiens misc_feature
(1)..(2790) Strandedness Double-stranded 5 atg ccc cgt gcc ccc cac
ttc atg ccc ttg ctg cta ctg ctg ctg ctg 48 Met Pro Arg Ala Pro His
Phe Met Pro Leu Leu Leu Leu Leu Leu Leu 1 5 10 15 ctc tca ctt ccc
cat act cag gcc gcc ttt ccc cag gac ccc ctc cct 96 Leu Ser Leu Pro
His Thr Gln Ala Ala Phe Pro Gln Asp Pro Leu Pro 20 25 30 ctg ttg
atc tct gac ctt caa ggt act tcc cca tta tcc tgg ttt cgg 144 Leu Leu
Ile Ser Asp Leu Gln Gly Thr Ser Pro Leu Ser Trp Phe Arg 35 40 45
ggc ctg gag gat gat gct gtg gct gca gaa ctt ggg ctg gac ttt cag 192
Gly Leu Glu Asp Asp Ala Val Ala Ala Glu Leu Gly Leu Asp Phe Gln 50
55 60 aga ttc ctg acc ttg aac cgg acc ttg cta gtg gct gcc cgg gat
cac 240 Arg Phe Leu Thr Leu Asn Arg Thr Leu Leu Val Ala Ala Arg Asp
His 65 70 75 80 gtt ttc tcc ttc gat ctt caa gcc gaa gaa gaa ggg gag
ggg ctg gtg 288 Val Phe Ser Phe Asp Leu Gln Ala Glu Glu Glu Gly Glu
Gly Leu Val 85 90 95 ccc aac aag tat cta aca tgg aga agc caa gat
gtg gag aac tgt gct 336 Pro Asn Lys Tyr Leu Thr Trp Arg Ser Gln Asp
Val Glu Asn Cys Ala 100 105 110 gta cgg gga aag ctg acg gat gag tgc
tac aac tat att cgt gtt ctt 384 Val Arg Gly Lys Leu Thr Asp Glu Cys
Tyr Asn Tyr Ile Arg Val Leu 115 120 125 gtt ccc tgg gac tcc cag acg
ctc ctt gcc tgt gga acg aac tca ttc 432 Val Pro Trp Asp Ser Gln Thr
Leu Leu Ala Cys Gly Thr Asn Ser Phe 130 135 140 agc cct gtg tgc cgc
agc tat ggg ata act tcg ctg cag cag gag ggt 480 Ser Pro Val Cys Arg
Ser Tyr Gly Ile Thr Ser Leu Gln Gln Glu Gly 145 150 155 160 gag gaa
ctg agt ggg cag gct cga tgc ccc ttt gat gcc acc cag tcc 528 Glu Glu
Leu Ser Gly Gln Ala Arg Cys Pro Phe Asp Ala Thr Gln Ser 165 170 175
aac gtg gcc atc ttt gca gag ggc agc ctg tac tca gcc aca gct gcg 576
Asn Val Ala Ile Phe Ala Glu Gly Ser Leu Tyr Ser Ala Thr Ala Ala 180
185 190 gat ttc cag gcc agt gat gct gta gtt tac aga agc ctt ggg ccc
cag 624 Asp Phe Gln Ala Ser Asp Ala Val Val Tyr Arg Ser Leu Gly Pro
Gln 195 200 205 ccc cca ctc cgc tcc gcc aag tat gac tcc aag tgg ctc
cga gag cca 672 Pro Pro Leu Arg Ser Ala Lys Tyr Asp Ser Lys Trp Leu
Arg Glu Pro 210 215 220 cac ttt gtc cag gcc ttg gag cat gga gac cat
gtc tac ttc ttc ttc 720 His Phe Val Gln Ala Leu Glu His Gly Asp His
Val Tyr Phe Phe Phe 225 230 235 240 cgc gag gtc tct gtg gag gat gct
cgg ctg ggg aag gtg cag ttc tcc 768 Arg Glu Val Ser Val Glu Asp Ala
Arg Leu Gly Lys Val Gln Phe Ser 245 250 255 cgc gta gcc cga gta tgt
aaa cgt gac atg ggc ggc tcg cct cgg gcc 816 Arg Val Ala Arg Val Cys
Lys Arg Asp Met Gly Gly Ser Pro Arg Ala 260 265 270 ttg gac cgc cac
tgg aca tcc ttc ctg aag ctt cgg ctc aac tgc tct 864 Leu Asp Arg His
Trp Thr Ser Phe Leu Lys Leu Arg Leu Asn Cys Ser 275 280 285 gtc cct
ggg gac tct act ttc tat ttt gat gtt tta cag gcc ttg act 912 Val Pro
Gly Asp Ser Thr Phe Tyr Phe Asp Val Leu Gln Ala Leu Thr 290 295 300
ggg cct gtg aac ctg cat ggc cgc tct gct ctc ttt ggg gtc ttc acc 960
Gly Pro Val Asn Leu His Gly Arg Ser Ala Leu Phe Gly Val Phe Thr 305
310 315 320 acc cag acc aat agc atc cct ggc tct gcc gtc tgc gcc ttc
tac ctg 1008 Thr Gln Thr Asn Ser Ile Pro Gly Ser Ala Val Cys Ala
Phe Tyr Leu 325 330 335 gat gag att gag cgt ggg ttt gag ggc aag ttc
aag gag cag agg agt 1056 Asp Glu Ile Glu Arg Gly Phe Glu Gly Lys
Phe Lys Glu Gln Arg Ser 340 345 350 ctg gat ggg gcc tgg act cct gtg
tct gag gac aga gtt ccc tca ccc 1104 Leu Asp Gly Ala Trp Thr Pro
Val Ser Glu Asp Arg Val Pro Ser Pro 355 360 365 agg cca gga tcc tgt
gca gga gta ggg gga gct gcc ttg ttc tcc tct 1152 Arg Pro Gly Ser
Cys Ala Gly Val Gly Gly Ala Ala Leu Phe Ser Ser 370 375 380 tcc cga
gac ctc cct gat gat gtc ctg acc ttc atc aag gct cac ccg 1200 Ser
Arg Asp Leu Pro Asp Asp Val Leu Thr Phe Ile Lys Ala His Pro 385 390
395 400 ctg ctg gac ccc gct gta cca cct gtc acc cat cag cct cta ctc
act 1248 Leu Leu Asp Pro Ala Val Pro Pro Val Thr His Gln Pro Leu
Leu Thr 405 410 415 ctc act agc agg gcc cta ctg acc caa gta gct gtg
gat ggc atg gct 1296 Leu Thr Ser Arg Ala Leu Leu Thr Gln Val Ala
Val Asp Gly Met Ala 420 425 430 ggt ccc cac agt aac atc aca gtc atg
ttc ctt ggc tcc aat gat ggg 1344 Gly Pro His Ser Asn Ile Thr Val
Met Phe Leu Gly Ser Asn Asp Gly 435 440 445 aca gtg ctg aag gtg ctg
acc cca ggt ggg cga tcc ggg gga cct gag 1392 Thr Val Leu Lys Val
Leu Thr Pro Gly Gly Arg Ser Gly Gly Pro Glu 450 455 460 ccc atc ctc
ctg gaa gag att gat gcc tac agc cct gcc cgg tgc agt 1440 Pro Ile
Leu Leu Glu Glu Ile Asp Ala Tyr Ser Pro Ala Arg Cys Ser 465 470 475
480 ggg aag cgg aca gcc caa aca gca cga cgg atc ata ggg ctg gag ctg
1488 Gly Lys Arg Thr Ala Gln Thr Ala Arg Arg Ile Ile Gly Leu Glu
Leu 485 490 495 gac act gag ggt cac agg ctt ttt gtg gct ttt tct ggc
tgt att gtc 1536 Asp Thr Glu Gly His Arg Leu Phe Val Ala Phe Ser
Gly Cys Ile Val 500 505 510 tac ctc cct ctc agc cgg tgt gcc cgg cat
ggg gcc tgt cag agg agc 1584 Tyr Leu Pro Leu Ser Arg Cys Ala Arg
His Gly Ala Cys Gln Arg Ser 515 520 525 tgt ttg gct tct cag gac cca
tac tgt gga tgg cat agc tcc agg ggc 1632 Cys Leu Ala Ser Gln Asp
Pro Tyr Cys Gly Trp His Ser Ser Arg Gly 530 535 540 tgt gtg gat atc
agg gga tct ggt ggg act gat gtg gat cag gct ggg 1680 Cys Val Asp
Ile Arg Gly Ser Gly Gly Thr Asp Val Asp Gln Ala Gly 545 550 555 560
aac cag gaa tcc atg gag cat ggt gac tgc caa gat gga gct act ggg
1728 Asn Gln Glu Ser Met Glu His Gly Asp Cys Gln Asp Gly Ala Thr
Gly 565 570 575 agt cag tct ggc cct ggg gat tct gct tat ggc gtg cgc
cgg gac ctg 1776 Ser Gln Ser Gly Pro Gly Asp Ser Ala Tyr Gly Val
Arg Arg Asp Leu 580 585 590 ccc cca gcc tcg gcc tcc cgc tcc gtc ccc
atc cca ctc ctc ctg gcc 1824 Pro Pro Ala Ser Ala Ser Arg Ser Val
Pro Ile Pro Leu Leu Leu Ala 595 600 605 agt gtg gcc gca gct ttt gcc
ctg ggc gcc tca gtc tct ggc ctc ctg 1872 Ser Val Ala Ala Ala Phe
Ala Leu Gly Ala Ser Val Ser Gly Leu Leu 610 615 620 gtc tcc tgt gct
tgt cgc cgc gcc cac cga cgt cgg ggc aag gac atc 1920 Val Ser Cys
Ala Cys Arg Arg Ala His Arg Arg Arg Gly Lys Asp Ile 625 630 635 640
gag act ccc ggg ctc ccg cgc cct ctc tcc ctc cgc agt ttg gcc cgg
1968 Glu Thr Pro Gly Leu Pro Arg Pro Leu Ser Leu Arg Ser Leu Ala
Arg 645 650 655 ctc cac ggt ggg ggc cca gag ccc ccg ccg ccc tcc aag
gac ggg gac 2016 Leu His Gly Gly Gly Pro Glu Pro Pro Pro Pro Ser
Lys Asp Gly Asp 660 665 670 gcg gtg cag acg ccg cag ctc tac acc acc
ttc ctg ccg cct ccg gag 2064 Ala Val Gln Thr Pro Gln Leu Tyr Thr
Thr Phe Leu Pro Pro Pro Glu 675 680 685 ggc gtg ccc ccg ccg gag ctg
gcc tgc ctg ccc acc ccc gag tcc acg 2112 Gly Val Pro Pro Pro Glu
Leu Ala Cys Leu Pro Thr Pro Glu Ser Thr 690 695 700 ccg gag ctg ccg
gtc aag cac ctc cgc gcc gcc ggg gac ccc tgg gag 2160 Pro Glu Leu
Pro Val Lys His Leu Arg Ala Ala Gly Asp Pro Trp Glu 705 710 715 720
tgg aac cag aac agg aac aac gcc aag gag ggt ccg ggc cgc tca cgg
2208 Trp Asn Gln Asn Arg Asn Asn Ala Lys Glu Gly Pro Gly Arg Ser
Arg 725 730 735 ggc ggg cac gcg gcg ggc ggg ccc gcg ccc cgc gtg ctg
gtg agg cca 2256 Gly Gly His Ala Ala Gly Gly Pro Ala Pro Arg Val
Leu Val Arg Pro 740 745 750 ccg ccg ccc ggc tgt ccc ggg cag gcc gtg
gaa gtc acc acc ctg gag 2304 Pro Pro Pro Gly Cys Pro Gly Gln Ala
Val Glu Val Thr Thr Leu Glu 755 760 765 gaa ctg ctg cgc tac ctg cac
ggc ccg cag ccg ccc aga aag ggg gcc 2352 Glu Leu Leu Arg Tyr Leu
His Gly Pro Gln Pro Pro Arg Lys Gly Ala 770 775 780 gag ccc ccc gcc
cct tta acc tcg cgg gcg ctc ccg ccg gag ccc gcc 2400 Glu Pro Pro
Ala Pro Leu Thr Ser Arg Ala Leu Pro Pro Glu Pro Ala 785 790 795 800
ccc gcc ctc ttg ggc ggc ccc agc ccc agg ccc cac gag tgc gcc tcg
2448 Pro Ala Leu Leu Gly Gly Pro Ser Pro Arg Pro His Glu Cys Ala
Ser 805 810 815 ccg
ctg agg ctg gac gtg ccc ccc gag ggc agg tgc gcc tct gcc ccc 2496
Pro Leu Arg Leu Asp Val Pro Pro Glu Gly Arg Cys Ala Ser Ala Pro 820
825 830 gcc cgg ccc gcg ctc tcc gcc ccc gct ccc cgg ctg ggc gtc ggc
gga 2544 Ala Arg Pro Ala Leu Ser Ala Pro Ala Pro Arg Leu Gly Val
Gly Gly 835 840 845 ggc cgg agg ttg cct ttc tcc ggc cac cgg gcc ccc
cct gcc ctg ctc 2592 Gly Arg Arg Leu Pro Phe Ser Gly His Arg Ala
Pro Pro Ala Leu Leu 850 855 860 act cga gtc ccc tcg gga ggt ccc tcc
agg tac tcc ggg ggt ccc ggg 2640 Thr Arg Val Pro Ser Gly Gly Pro
Ser Arg Tyr Ser Gly Gly Pro Gly 865 870 875 880 aag cac ctc ctg tac
ctg ggc cgg ccc gag ggc tac cgg ggc cgc gcc 2688 Lys His Leu Leu
Tyr Leu Gly Arg Pro Glu Gly Tyr Arg Gly Arg Ala 885 890 895 ctg aaa
agg gtg gac gtc gag aag ccc cag ttg tcc ctg aag cct ccc 2736 Leu
Lys Arg Val Asp Val Glu Lys Pro Gln Leu Ser Leu Lys Pro Pro 900 905
910 ctc gtc ggg ccc tcc tcc cgc cag gcc gtc ccg aac ggc ggc cgt ttc
2784 Leu Val Gly Pro Ser Ser Arg Gln Ala Val Pro Asn Gly Gly Arg
Phe 915 920 925 aac ttt 2790 Asn Phe 930 6 930 PRT Homo sapiens
misc_feature ()..() Tissue Type Child Brain 6 Met Pro Arg Ala Pro
His Phe Met Pro Leu Leu Leu Leu Leu Leu Leu 1 5 10 15 Leu Ser Leu
Pro His Thr Gln Ala Ala Phe Pro Gln Asp Pro Leu Pro 20 25 30 Leu
Leu Ile Ser Asp Leu Gln Gly Thr Ser Pro Leu Ser Trp Phe Arg 35 40
45 Gly Leu Glu Asp Asp Ala Val Ala Ala Glu Leu Gly Leu Asp Phe Gln
50 55 60 Arg Phe Leu Thr Leu Asn Arg Thr Leu Leu Val Ala Ala Arg
Asp His 65 70 75 80 Val Phe Ser Phe Asp Leu Gln Ala Glu Glu Glu Gly
Glu Gly Leu Val 85 90 95 Pro Asn Lys Tyr Leu Thr Trp Arg Ser Gln
Asp Val Glu Asn Cys Ala 100 105 110 Val Arg Gly Lys Leu Thr Asp Glu
Cys Tyr Asn Tyr Ile Arg Val Leu 115 120 125 Val Pro Trp Asp Ser Gln
Thr Leu Leu Ala Cys Gly Thr Asn Ser Phe 130 135 140 Ser Pro Val Cys
Arg Ser Tyr Gly Ile Thr Ser Leu Gln Gln Glu Gly 145 150 155 160 Glu
Glu Leu Ser Gly Gln Ala Arg Cys Pro Phe Asp Ala Thr Gln Ser 165 170
175 Asn Val Ala Ile Phe Ala Glu Gly Ser Leu Tyr Ser Ala Thr Ala Ala
180 185 190 Asp Phe Gln Ala Ser Asp Ala Val Val Tyr Arg Ser Leu Gly
Pro Gln 195 200 205 Pro Pro Leu Arg Ser Ala Lys Tyr Asp Ser Lys Trp
Leu Arg Glu Pro 210 215 220 His Phe Val Gln Ala Leu Glu His Gly Asp
His Val Tyr Phe Phe Phe 225 230 235 240 Arg Glu Val Ser Val Glu Asp
Ala Arg Leu Gly Lys Val Gln Phe Ser 245 250 255 Arg Val Ala Arg Val
Cys Lys Arg Asp Met Gly Gly Ser Pro Arg Ala 260 265 270 Leu Asp Arg
His Trp Thr Ser Phe Leu Lys Leu Arg Leu Asn Cys Ser 275 280 285 Val
Pro Gly Asp Ser Thr Phe Tyr Phe Asp Val Leu Gln Ala Leu Thr 290 295
300 Gly Pro Val Asn Leu His Gly Arg Ser Ala Leu Phe Gly Val Phe Thr
305 310 315 320 Thr Gln Thr Asn Ser Ile Pro Gly Ser Ala Val Cys Ala
Phe Tyr Leu 325 330 335 Asp Glu Ile Glu Arg Gly Phe Glu Gly Lys Phe
Lys Glu Gln Arg Ser 340 345 350 Leu Asp Gly Ala Trp Thr Pro Val Ser
Glu Asp Arg Val Pro Ser Pro 355 360 365 Arg Pro Gly Ser Cys Ala Gly
Val Gly Gly Ala Ala Leu Phe Ser Ser 370 375 380 Ser Arg Asp Leu Pro
Asp Asp Val Leu Thr Phe Ile Lys Ala His Pro 385 390 395 400 Leu Leu
Asp Pro Ala Val Pro Pro Val Thr His Gln Pro Leu Leu Thr 405 410 415
Leu Thr Ser Arg Ala Leu Leu Thr Gln Val Ala Val Asp Gly Met Ala 420
425 430 Gly Pro His Ser Asn Ile Thr Val Met Phe Leu Gly Ser Asn Asp
Gly 435 440 445 Thr Val Leu Lys Val Leu Thr Pro Gly Gly Arg Ser Gly
Gly Pro Glu 450 455 460 Pro Ile Leu Leu Glu Glu Ile Asp Ala Tyr Ser
Pro Ala Arg Cys Ser 465 470 475 480 Gly Lys Arg Thr Ala Gln Thr Ala
Arg Arg Ile Ile Gly Leu Glu Leu 485 490 495 Asp Thr Glu Gly His Arg
Leu Phe Val Ala Phe Ser Gly Cys Ile Val 500 505 510 Tyr Leu Pro Leu
Ser Arg Cys Ala Arg His Gly Ala Cys Gln Arg Ser 515 520 525 Cys Leu
Ala Ser Gln Asp Pro Tyr Cys Gly Trp His Ser Ser Arg Gly 530 535 540
Cys Val Asp Ile Arg Gly Ser Gly Gly Thr Asp Val Asp Gln Ala Gly 545
550 555 560 Asn Gln Glu Ser Met Glu His Gly Asp Cys Gln Asp Gly Ala
Thr Gly 565 570 575 Ser Gln Ser Gly Pro Gly Asp Ser Ala Tyr Gly Val
Arg Arg Asp Leu 580 585 590 Pro Pro Ala Ser Ala Ser Arg Ser Val Pro
Ile Pro Leu Leu Leu Ala 595 600 605 Ser Val Ala Ala Ala Phe Ala Leu
Gly Ala Ser Val Ser Gly Leu Leu 610 615 620 Val Ser Cys Ala Cys Arg
Arg Ala His Arg Arg Arg Gly Lys Asp Ile 625 630 635 640 Glu Thr Pro
Gly Leu Pro Arg Pro Leu Ser Leu Arg Ser Leu Ala Arg 645 650 655 Leu
His Gly Gly Gly Pro Glu Pro Pro Pro Pro Ser Lys Asp Gly Asp 660 665
670 Ala Val Gln Thr Pro Gln Leu Tyr Thr Thr Phe Leu Pro Pro Pro Glu
675 680 685 Gly Val Pro Pro Pro Glu Leu Ala Cys Leu Pro Thr Pro Glu
Ser Thr 690 695 700 Pro Glu Leu Pro Val Lys His Leu Arg Ala Ala Gly
Asp Pro Trp Glu 705 710 715 720 Trp Asn Gln Asn Arg Asn Asn Ala Lys
Glu Gly Pro Gly Arg Ser Arg 725 730 735 Gly Gly His Ala Ala Gly Gly
Pro Ala Pro Arg Val Leu Val Arg Pro 740 745 750 Pro Pro Pro Gly Cys
Pro Gly Gln Ala Val Glu Val Thr Thr Leu Glu 755 760 765 Glu Leu Leu
Arg Tyr Leu His Gly Pro Gln Pro Pro Arg Lys Gly Ala 770 775 780 Glu
Pro Pro Ala Pro Leu Thr Ser Arg Ala Leu Pro Pro Glu Pro Ala 785 790
795 800 Pro Ala Leu Leu Gly Gly Pro Ser Pro Arg Pro His Glu Cys Ala
Ser 805 810 815 Pro Leu Arg Leu Asp Val Pro Pro Glu Gly Arg Cys Ala
Ser Ala Pro 820 825 830 Ala Arg Pro Ala Leu Ser Ala Pro Ala Pro Arg
Leu Gly Val Gly Gly 835 840 845 Gly Arg Arg Leu Pro Phe Ser Gly His
Arg Ala Pro Pro Ala Leu Leu 850 855 860 Thr Arg Val Pro Ser Gly Gly
Pro Ser Arg Tyr Ser Gly Gly Pro Gly 865 870 875 880 Lys His Leu Leu
Tyr Leu Gly Arg Pro Glu Gly Tyr Arg Gly Arg Ala 885 890 895 Leu Lys
Arg Val Asp Val Glu Lys Pro Gln Leu Ser Leu Lys Pro Pro 900 905 910
Leu Val Gly Pro Ser Ser Arg Gln Ala Val Pro Asn Gly Gly Arg Phe 915
920 925 Asn Phe 930 7 170 DNA Homo sapiens misc_feature (1)..(170)
Strandedness Double-stranded 7 tggctgtatt gtctacctcc ctctcagccg
gtgtgcccgg catggggcct gtcagaggag 60 ctgtttggct tctcaggacc
catactgtgg atggcatagc tccaggggct gtgtggatat 120 caggggatct
ggtgggactg atgtggatca ggctnggaac caggaatcca 170 8 18 DNA Synthetic
DNA misc_feature (1)..(18) CDS; Identification Method P 8
tggctgtatt gtctacct 18 9 20 DNA Synthetic DNA misc_feature
(1)..(20) CDS; Identification Method P 9 tggattcctg gttccnagcc 20
10 18 DNA Synthetic DNA misc_feature (1)..(18) CDS; Identification
Method E 10 tgtgtaaacg tgacatgg 18 11 18 DNA Synthetic DNA
misc_feature (1)..(18) CDS; Identification Method E 11 tgctagtcag
agtgagga 18 12 7 PRT Homo sapiens misc_feature (1)..(1) First "Xaa"
is Gln or Arg 12 Xaa Asp Pro Tyr Cys Xaa Trp 1 5 13 14 PRT Unknown
misc_feature ()..() Description of Unknown Organism Myc tag 13 Asp
Ile Gly Gly Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu 1 5 10
* * * * *