U.S. patent application number 10/792481 was filed with the patent office on 2004-08-05 for immediate early genes and methods of use therefor.
This patent application is currently assigned to THE JOHNS HOPKINS UNIVERSITY SCHOOL OF MEDICINE. Invention is credited to Goetz, Bernhard, Hiemisch, Holger, Kuner, Rohini, Lanahan, Anthony, Nikolich, Karoly, Scheek, Sigrid, Worley, Paul F., Zhukovsky, Eugene.
Application Number | 20040152658 10/792481 |
Document ID | / |
Family ID | 26755291 |
Filed Date | 2004-08-05 |
United States Patent
Application |
20040152658 |
Kind Code |
A1 |
Worley, Paul F. ; et
al. |
August 5, 2004 |
Immediate early genes and methods of use therefor
Abstract
The present invention provides methods and materials related to
immediate early genes. Specifically, the invention provides
isolated immediate early gene nucleic acid, cells that contain
isolated immediate early gene nucleic acid, substantially pure
polypeptides encoded by immediate early gene nucleic acid, and
antibodies having specific binding affinity for a polypeptide
encoded by immediate early gene nucleic acid. In addition, the
invention provides cDNA libraries enriched for immediate early
genes cDNAs, isolated nucleic acid derived from such cDNA
libraries, and methods for treating conditions related to a
deficiency in a neuron's immediate early gene responsiveness to a
stimulus.
Inventors: |
Worley, Paul F.; (Baltimore,
MD) ; Lanahan, Anthony; (Baltimore, MD) ;
Goetz, Bernhard; (Heidelberg, DE) ; Hiemisch,
Holger; (Heidelberg, DE) ; Kuner, Rohini;
(Heidelberg, DE) ; Scheek, Sigrid; (Dossenheim,
DE) ; Nikolich, Karoly; (Redwood City, CA) ;
Zhukovsky, Eugene; (San Francisco, CA) |
Correspondence
Address: |
Lisa A. Haile, J.D., Ph.D.
GRAY CARY WARE & FREIDENRICH LLP
Suite 1100
4365 Executive Drive
San Diego
CA
92121-2133
US
|
Assignee: |
THE JOHNS HOPKINS UNIVERSITY SCHOOL
OF MEDICINE
BASF-LYNX BIOSCIENCE AG
LYNX THERAPEUTICS, INC.
|
Family ID: |
26755291 |
Appl. No.: |
10/792481 |
Filed: |
March 2, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10792481 |
Mar 2, 2004 |
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09244805 |
Feb 5, 1999 |
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6699660 |
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60074518 |
Feb 12, 1998 |
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60074135 |
Feb 9, 1998 |
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Current U.S.
Class: |
514/44R ;
435/325; 536/23.1 |
Current CPC
Class: |
C12Q 1/6883 20130101;
C12Q 2600/158 20130101; A61P 25/00 20180101; C07K 14/47 20130101;
A61K 38/00 20130101; A61P 25/28 20180101; C12Q 1/6876 20130101;
C12Q 2600/136 20130101 |
Class at
Publication: |
514/044 ;
435/006; 536/023.1 |
International
Class: |
A61K 048/00; C12Q
001/68; C07H 021/04 |
Goverment Interests
[0002] Funding for the work described herein was provided by the
federal government, which may have certain rights in the invention.
Claims
What is claimed is:
1. An isolated nucleic acid comprising at least one adenine base,
at least one guanine base, at least one cytosine base, and at least
one thymine or uracil base, wherein said isolated nucleic acid is
at least 12 bases in length, and hybridizes to the sense or
antisense strand of a second nucleic acid under hybridization
conditions, said second nucleic acid having a sequence as set forth
in SEQ ID NO:3, 4, 12, 18, 19, 26, 31, 45, 46, 58, 59, or 60.
2. The isolated nucleic acid of claim 1, wherein said hybridization
conditions are moderately stringent hybridization conditions.
3. The isolated nucleic acid of claim 1, wherein said hybridization
conditions are highly stringent hybridization conditions.
4. An isolated nucleic acid, wherein said isolated nucleic acid
comprises a nucleic acid sequence that encodes an amino acid
sequence at least five amino acids in length, said amino acid
sequence comprising at least three different amino acid residues,
and being identical to a contiguous portion of sequence set forth
in SEQ ID NO:27, 32, 61, or 62.
5. An isolated nucleic acid comprising a nucleic acid sequence at
least 60 percent identical to the sequence set forth in SEQ ID
NO:3, 4, 12, 18, 19, 26, 31, 45, 46, 58, 59, or 60.
6. An isolated nucleic acid, wherein said isolated nucleic acid
comprises a nucleic acid sequence that encodes an amino acid
sequence at least 60 percent identical to the sequence set forth in
SEQ ID NO:27, 32, 61, or 62.
7. An isolated nucleic acid comprising a nucleic acid sequence as
set forth in SEQ ID NO:3, 4,12, 18, 19, 26, 31, 45, 46, 58, 59, or
60.
8. A substantially pure polypeptide comprising an amino acid
sequence encoded by a nucleic acid of claim 1.
9. A substantially pure polypeptide comprising an amino acid
sequence as set forth in SEQ ID NO:27, 32, 61, or 62.
10. A substantially pure polypeptide comprising an amino acid
sequence at least 60 percent identical to the sequence set forth in
SEQ ID NO:27, 32, 61, or 62.
11. A substantially pure polypeptide comprising an amino acid
sequence at least five amino acids in length, said amino acid
sequence comprising at least three different amino acid residues,
and being identical to a contiguous stretch of sequence set forth
in SEQ ID NO:27, 32, 61, or 62.
12. A host cell containing an isolated nucleic acid of claim 1.
13. The host cell of claim 12, wherein said host cell is a
eukaryotic cell.
14. An antibody having specific binding affinity for an amino acid
sequence encoded by a nucleic acid of claim 1.
15. The antibody of claim 14, wherein said antibody is
monoclonal.
16. The antibody of claim 14, wherein said antibody is
polyclonal.
17. A cDNA library comprising a plurality of clones, wherein each
clone comprises a cDNA insert and wherein at least about 15 percent
of said clones comprise cDNA derived from immediate early
genes.
18. The cDNA library of claim 17, wherein at least about 20 percent
of said clones comprise cDNA derived from immediate early
genes.
19. The cDNA library of claim 17, wherein at least about 25 percent
of said clones comprise cDNA derived from immediate early
genes.
20. The cDNA library of claim 17, wherein said immediate early
genes are immediate early genes responsive to a maximal
electroconvulsive seizure.
21. The cDNA library of claim 17, wherein said cDNA library is a
subtracted cDNA library.
22. The cDNA library of claim 21, wherein said subtracted cDNA
library is IEG-Reg cDNA library.
23. The cDNA library of claim 21, wherein said subtracted cDNA
library is IEG-Lg cDNA library.
24. An isolated nucleic acid derived from a cDNA library, wherein
said cDNA library comprises a plurality of clones, wherein each
clone comprises a cDNA insert and wherein at least about 15 percent
of said clones comprise cDNA derived from immediate early
genes.
25. The isolated nucleic acid of claim 24, wherein said isolated
nucleic acid comprises a nucleic acid sequence of an immediate
early gene.
26. A method of obtaining immediate early gene nucleic acid, said
method comprising: a) providing a cDNA library, said cDNA library
comprising a plurality of clones, wherein each clone comprises a
cDNA insert and wherein at least about 15 percent of said clones
comprise cDNA derived from immediate early genes; b) contacting at
least a portion of said cDNA library with a probe, said probe
containing at least one nucleic acid having a nucleic acid sequence
derived from an immediate early gene; and c) selecting a member of
said plurality of clones based on the hybridization of said at
least one nucleic acid to said member under hybridization
conditions, said member comprising said immediate early gene
nucleic acid.
27. A method of treating an animal having a deficiency in a
neuron's immediate early gene responsiveness to a stimulus, said
method comprising administering a nucleic acid of claim 1 to said
animal such that the effect of said deficiency is minimized.
28. The method of claim 27, wherein said deficiency comprises a
reduced level of expression of an immediate early gene.
29. The method of claim 27, wherein said stimulus influences
learning or memory.
30. The method of claim 29, wherein said stimulus comprises a
maximal electroconvulsive seizure.
31. A method of treating an animal having a deficiency in a
neuron's immediate early gene responsiveness to a stimulus, said
method comprising administering a therapeutically effective amount
of a substantially pure polypeptide of claim 8 to said animal such
that the effect of said deficiency is minimized.
32. A method of treating an animal having a deficiency in a
neuron's immediate early gene responsiveness to a stimulus, said
method comprising administering an effective amount of cells to
said animal such that the effect of said deficiency is minimized,
said cells containing a nucleic acid of claim 1.
33. A method of treating an animal having a deficiency in a
neuron's immediate early gene responsiveness to a stimulus, said
method comprising administering a therapeutically effective of
antibodies to said animal such that the effect of said deficiency
is minimized, said antibodies having specific binding affinity for
an amino acid sequence encoded by a nucleic acid of claim 1.
34. The method of claim 33, wherein said deficiency comprises an
elevated level of expression of an immediate early gene.
35. A method of identifying a compound that modulates immediate
early gene expression, said method comprising: a) contacting a test
compound with an immediate early gene nucleic acid; and b)
determining whether said test compound effects the expression of
said immediate early gene nucleic acid, wherein the presence of an
effect indicates that said test compound is said compound.
36. The method of claim 35, wherein said immediate early gene
nucleic acid comprises a nucleic acid sequence as set forth in SEQ
ID NO:3, 4, 12, 18, 19, 26, 31, 45, 46, 58, 59, or 60.
37. The method of claim 35, wherein said effect is a reduction in
the expression of said immediate early gene nucleic acid.
38. The method of claim 35, wherein said effect is an increase in
the expression of said immediate early gene nucleic acid.
39. A method of identifying a compound that modulates immediate
early gene polypeptide activity, said method comprising: a)
contacting a test compound with an immediate early gene
polypeptide; and b) determining whether said test compound effects
the activity of said immediate early gene polypeptide, wherein the
presence of an effect indicates that said test compound is said
compound.
40. The method of claim 39, wherein said immediate early gene
polypeptide comprises an amino acid sequence encoded by a nucleic
acid of claim 1.
41. The method of claim 39, wherein said immediate early gene
polypeptide comprises an amino acid sequence as set forth in SEQ ID
NO:27, 32, 61, or 62.
42. The method of claim 39, wherein said effect is a reduction in
the activity of said immediate early gene polypeptide.
43. The method of claim 39, wherein said effect is an increase in
the activity of said immediate early gene polypeptide.
Description
RELATED APPLICATIONS
[0001] This application claims priority to U.S. provisional
application Nos. 60/074,518, filed Feb. 12, 1998 and 60/074,135,
filed Feb. 6, 1998, both of which are incorporated herein by
reference.
BACKGROUND
[0003] 1. Technical Field
[0004] The present invention generally relates to gene expression
and more specifically to immediate early genes in the brain and
polypeptides encoded by such immediate early genes.
[0005] 2. Background Information
[0006] An immediate early gene (IEG) is a gene whose expression is
rapidly increased immediately following a stimulus. For example,
genes expressed by neurons that exhibit a rapid increase in
expression immediately following neuronal stimulation are neuronal
IEGs. Such neuronal IEGs have been found to encode a wide variety
of polypeptides including transcription factors, cytoskeletal
polypeptides, growth factors, and metabolic enzymes as well as
polypeptides involved in signal transduction. The identification of
neuronal IEGs and the polypeptides they encode provides important
information about the function of neurons in, for example,
learning, memory, synaptic transmission, tolerance, and neuronal
plasticity.
SUMMARY
[0007] The present invention involves methods and materials related
to IEGs. Specifically, the invention provides isolated IEG nucleic
acid sequences, cells that contain isolated IEG nucleic acid,
substantially pure polypeptides encoded by IEG nucleic acid, and
antibodies having specific binding affinity for a polypeptide
encoded by IEG nucleic acid. In addition, the invention provides
cDNA libraries enriched for IEG cDNAs, isolated nucleic acid
derived from such cDNA libraries, and methods for treating
conditions related to a deficiency in a neuron's IEG responsiveness
to a stimulus.
[0008] In one aspect, the invention features an isolated nucleic
acid having at least one adenine base, at least one guanine base,
at least one cytosine base, and at least one thymine or uracil
base. The isolated nucleic acid is at least 12 bases in length, and
hybridizes to the sense or antisense strand of a second nucleic
acid under hybridization conditions. The second nucleic acid has a
sequence as set forth in SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 23, 24, 25, 26, 28, 29, 31,
33, 34, 35, 37, 39, 40, 41, 42, 43, 44, 45, 46, 47, 49, 50, 51, 52,
53, 54, 55, 56, 57, 58, 59, or 60. The hybridization conditions can
be moderately or highly stringent hybridization conditions.
[0009] In another embodiment, the invention features an isolated
nucleic acid having a nucleic acid sequence that encodes an amino
acid sequence at least five amino acids in length. The amino acid
sequence contains at least three different amino acid residues, and
is identical to a contiguous portion of sequence set forth in SEQ
ID NO:11, 21, 27, 30, 32, 36, 38, 48, 61, or 62.
[0010] Another embodiment of the invention features an isolated
nucleic acid having a nucleic acid sequence at least 60 percent
identical to the sequence set forth in SEQ ID NO:1, 2, 3, 4, 5, 6,
7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 23, 24, 25,
26, 28, 29, 31, 33, 34, 35, 37, 39, 40, 41, 42, 43, 44, 45, 46, 47,
49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60.
[0011] Another embodiment of the invention features an isolated
nucleic acid having a nucleic acid sequence that encodes an amino
acid sequence at least 60 percent identical to the sequence set
forth in SEQ ID NO:11, 21, 27, 30, 32, 36, 38, 48, 61, or 62.
[0012] Another embodiment of the invention features an isolated
nucleic acid having a nucleic acid sequence as set forth in SEQ ID
NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 18, 19,
20, 22, 23, 24, 25, 26, 28, 29, 31, 33, 34, 35, 37, 39, 40, 41, 42,
43, 44, 45, 46, 47, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or
60.
[0013] In another aspect, the invention features a substantially
pure polypeptide having an amino acid sequence encoded by a nucleic
acid having at least one adenine base, at least one guanine base,
at least one cytosine base, and at least one thymine or uracil
base. The nucleic acid is at least 12 bases in length, and
hybridizes to the sense or antisense strand of a second nucleic
acid under hybridization conditions. The second nucleic acid has a
sequence as set forth in SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 23, 24, 25, 26, 28, 29, 31,
33, 34, 35, 37, 39, 40, 41, 42, 43, 44, 45, 46, 47, 49, 50, 51, 52,
53, 54, 55, 56, 57, 58, 59, or 60.
[0014] In another embodiment, the invention features a
substantially pure polypeptide having an amino acid sequence as set
forth in SEQ ID NO:11, 21, 27, 30, 32, 36, 38, 48, 61, or 62.
[0015] Another embodiment of the invention features a substantially
pure polypeptide having an amino acid sequence at least 60 percent
identical to the sequence set forth in SEQ ID NO:11, 21, 27, 30,
32, 36, 38, 48, 61, or 62.
[0016] Another embodiment of the invention features a substantially
pure polypeptide having an amino acid sequence at least five amino
acids in length. The amino acid sequence contains at least three
different amino acid residues, and is identical to a contiguous
stretch of sequence set forth in SEQ ID NO:11, 21, 27, 30, 32, 36,
38, 48, 61, or 62.
[0017] Another aspect of the invention features a host cell (e.g.,
a eukaryotic or prokaryotic cell) containing an isolated nucleic
acid having at least one adenine base, at least one guanine base,
at least one cytosine base, and at least one thymine or uracil
base. The isolated nucleic acid is at least 12 bases in length, and
hybridizes to the sense or antisense strand of a second nucleic
acid under hybridization conditions. The second nucleic acid has a
sequence as set forth in SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 23, 24, 25, 26, 28, 29, 31,
33, 34, 35, 37, 39, 40, 41, 42, 43, 44, 45, 46, 47, 49, 50, 51, 52,
53, 54, 55, 56, 57, 58, 59, or 60.
[0018] Another aspect of the invention features an antibody (e.g.,
a monoclonal or polyclonal antibody) having specific binding
affinity for an amino acid sequence encoded by a nucleic acid
having at least one adenine base, at least one guanine base, at
least one cytosine base, and at least one thymine or uracil base.
The nucleic acid is at least 12 bases in length, and hybridizes to
the sense or antisense strand of a second nucleic acid under
hybridization conditions. The second nucleic acid has a sequence as
set forth in SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14,
15, 16, 17, 18, 19, 20, 22, 23, 24, 25, 26, 28, 29, 31, 33, 34, 35,
37, 39, 40, 41, 42, 43, 44, 45, 46, 47, 49, 50, 51, 52, 53, 54, 55,
56, 57, 58, 59, or 60.
[0019] Another aspect of the invention features a cDNA library
having a plurality of clones with each clone having a cDNA insert.
In addition, at least about 15 percent (e.g., at least about 20 or
25 percent) of the clones have cDNA derived from immediate early
genes (e.g., immediate early genes responsive to a maximal
electroconvulsive seizure). The cDNA library can be a subtracted
cDNA library. For example, the subtracted cDNA library can be the
IEG-Reg or IEG-Lg cDNA library.
[0020] Another aspect of the invention features an isolated nucleic
acid derived from a cDNA library. The cDNA library has a plurality
of clones with each clone having a cDNA insert. In addition, at
least about 15 percent of the clones have cDNA derived from
immediate early genes. The isolated nucleic acid can have a nucleic
acid sequence of an immediate early gene.
[0021] Another aspect of the invention features a method of
obtaining immediate early gene nucleic acid. The method includes
providing a cDNA library having a plurality of clones with each
clone having a cDNA insert. In addition, at least about 15 percent
of the clones have cDNA derived from immediate early genes. The
method also includes contacting at least a portion of the cDNA
library with a probe containing at least one nucleic acid having a
nucleic acid sequence derived from an immediate early gene, and
selecting a member of the plurality of clones based on the
hybridization of the at least one nucleic acid to the member under
hybridization conditions.
[0022] Another aspect of the invention features a method of
treating an animal (e.g., human) having a deficiency in a neuron's
immediate early gene responsiveness to a stimulus. The method
includes administering a nucleic acid to the animal such that the
effect of the deficiency is minimized. The nucleic acid has at
least one adenine base, at least one guanine base, at least one
cytosine base, and at least one thymine or uracil base. In
addition, the nucleic acid is at least 12 bases in length, and
hybridizes to the sense or antisense strand of a second nucleic
acid under hybridization conditions. The second nucleic acid has a
sequence as set forth in SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 23, 24, 25, 26, 28, 29, 31,
33, 34, 35, 37, 39, 40, 41, 42, 43, 44, 45, 46, 47, 49, 50, 51, 52,
53, 54, 55, 56, 57, 58, 59, or 60. The deficiency can include a
reduced level of expression of an immediate early gene. In
addition, the stimulus can influence learning or memory. For
example, the stimulus can include a maximal electroconvulsive
seizure.
[0023] In another embodiment, the invention features a method of
treating an animal (e.g., human) having a deficiency in a neuron's
immediate early gene responsiveness to a stimulus. The method
includes administering a therapeutically effective amount of a
substantially pure polypeptide to the animal such that the effect
of the deficiency is minimized. The polypeptide contains an amino
acid sequence encoded by a nucleic acid having at least one adenine
base, at least one guanine base, at least one cytosine base, and at
least one thymine or uracil base. The nucleic acid is at least 12
bases in length, and hybridizes to the sense or antisense strand of
a second nucleic acid under hybridization conditions. The second
nucleic acid has a sequence as set forth in SEQ ID NO:1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 23, 24,
25, 26, 28, 29, 31, 33, 34, 35, 37, 39, 40, 41, 42, 43, 44, 45, 46,
47, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60.
[0024] Another embodiment of the invention features a method of
treating an animal (e.g., human) having a deficiency in a neuron's
immediate early gene responsiveness to a stimulus. The method
includes administering an effective amount of cells to the animal
such that the effect of the deficiency is minimized. The cells
contain a nucleic acid having at least one adenine base, at least
one guanine base, at least one cytosine base, and at least one
thymine or uracil base. In addition, the nucleic acid is at least
12 bases in length, and hybridizes to the sense or antisense strand
of a second nucleic acid under hybridization conditions. The second
nucleic acid has a sequence as set forth in SEQ ID NO:1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 18, 19,20, 22, 23, 24,
25, 26, 28, 29, 31, 33, 34, 35, 37, 39, 40, 41, 42, 43, 44, 45, 46,
47, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60.
[0025] Another embodiment of the invention features a method of
treating an animal (e.g., human) having a deficiency in a neuron's
immediate early gene responsiveness to a stimulus. The method
includes administering a therapeutically effective of antibodies to
the animal such that the effect of the deficiency is minimized. The
antibodies have specific binding affinity for an amino acid
sequence encoded by a nucleic acid having at least one adenine
base, at least one guanine base, at least one cytosine base, and at
least one thymine or uracil base. The nucleic acid is at least 12
bases in length, and hybridizes to the sense or antisense strand of
a second nucleic acid under hybridization conditions. The second
nucleic acid has a sequence as set forth in SEQ ID NO:1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 23, 24,
25, 26, 28, 29, 31, 33, 34, 35, 37, 39, 40, 41, 42, 43, 44, 45, 46,
47, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60. The
deficiency can include an elevated level of expression of an
immediate early gene.
[0026] Another aspect of the invention features a method of
identifying a compound that modulates immediate early gene
expression. The method includes contacting a test compound with an
immediate early gene nucleic acid, and determining whether the test
compound effects the expression of the immediate early gene nucleic
acid. The presence of an effect indicates that the test compound is
a compound that modulates immediate early gene expression. The
immediate early gene nucleic acid can contain a nucleic acid
sequence as set forth in SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 23, 24, 25, 26, 28, 29, 31,
33, 34, 35, 37, 39, 40, 41, 42, 43, 44, 45, 46, 47, 49, 50, 51, 52,
53, 54, 55, 56, 57, 58, 59, or 60. The effect can be a reduction or
increase in the expression of the immediate early gene nucleic
acid.
[0027] In another embodiment, the invention features a method of
identifying a compound that modulates immediate early gene
polypeptide activity. The method includes contacting a test
compound with an immediate early gene polypeptide, and determining
whether the test compound effects the activity of the immediate
early gene polypeptide. The presence of an effect indicates that
the test compound is a compound that modulates immediate early gene
polypeptide activity. The immediate early gene polypeptide can
contain an amino acid sequence encoded by a nucleic acid having at
least one adenine base, at least one guanine base, at least one
cytosine base, and at least one thymine or uracil base. The nucleic
acid is at least 12 bases in length, and hybridizes to the sense or
antisense strand of a second nucleic acid under hybridization
conditions. The second nucleic acid has a sequence as set forth in
SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17,
18, 19, 20, 22, 23, 24, 25, 26, 28, 29, 31, 33, 34, 35, 37, 39, 40,
41, 42, 43, 44, 45, 46, 47, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,
59, or 60. Alternatively, the immediate early gene polypeptide can
contain an amino acid sequence as set forth in SEQ ID NO:11, 21,
27, 30, 32, 36, 38, 48, 61, or 62. The effect can be a reduction or
increase in the activity of the immediate early gene
polypeptide.
[0028] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention pertains.
Although methods and materials similar or equivalent to those
described herein can be used in the practice or testing of the
present invention, suitable methods and materials are described
below. All publications, patent applications, patents, and other
references mentioned herein are incorporated by reference in their
entirety. In case of conflict, the present specification, including
definitions, will control. In addition, the materials, methods, and
examples are illustrative only and not intended to be limiting.
[0029] Other features and advantages of the invention will be
apparent from the following detailed description, and from the
claims.
DETAILED DESCRIPTION
[0030] The present invention provides methods and materials related
to EEGs. Specifically, the invention provides isolated IEG nucleic
acid, cells that contain isolated IEG nucleic acid, substantially
pure polypeptides encoded by IEG nucleic acid, and antibodies
having specific binding affinity for a polypeptide encoded by IEG
nucleic acid. In addition, the invention provides cDNA libraries
enriched for IEG cDNAs, isolated nucleic acid derived from such
cDNA libraries, and methods for treating conditions related to a
deficiency in a neuron's IEG responsiveness to a stimulus.
[0031] The present invention is based on the discovery of nucleic
acid clones for many different neuronal IEGs. Specifically, nucleic
acid clones for different neuronal IEGs were isolated and
identified based on the ability of each IEG to rapidly increase
expression upon seizure induction by a maximal electroconvulsive
seizure (MECS) method (Cole et al., J. Neurochem. 55:1920-1927
(1990)). It is important to note that MECS induction can be
considered a model to study long-term plasticity relevant to
learning and memory since it is known that a single MECS can
produce extremely robust and long lived potentiation of synaptic
contacts in the hippocampus and block spatial learning (Barnes et
al., J. Neurosci. 14:5793-5806 (1994)). Thus, MECS-responsive IEGs
can influence neuronal activities involved in brain functions such
as learning and memory. Moreover, the isolation and identification
of IEG nucleic acid not only provides research scientists with
information about neuronal activity and gene regulation but also
provides methods and materials that can be used to manipulate brain
function.
[0032] Each isolated IEG nucleic acid described herein can be used
to produce a polypeptide. In addition, each IEG nucleic acid can be
used to identify cells that are responsive to MECS induction. For
example, an EEG nucleic acid can be labeled and used as a probe for
in situ hybridization analysis. Clearly, having the ability to
identify MECS-responsive cells provides one with the ability to
isolate or monitor specific brain regions that are involved in
learning. Further, any of the isolated partial IEG nucleic acid
sequences can be used to obtain a full-length clone that encodes an
IEG polypeptide. For example, a fragment from an isolated IEG
nucleic acid can be radioactively labeled and used to screen a
library such that a full-length clone is obtained.
[0033] Cells containing isolated IEG nucleic acid can be used to
maintain or propagate the isolated IEG nucleic acid. In addition,
such cells can be used to produce large quantities of polypeptides
that are encoded by isolated IEG nucleic acid. Further, cells
containing isolated IEG nucleic acid can be used to generate virus
particles containing the isolated IEG nucleic acid. Such virus
particles can be used in vitro or in vivo to provide other cells
with the isolated IEG nucleic acid. The polypeptides encoded by IEG
nucleic acid can be used as immunogens to produce antibodies. Such
antibodies can be used to identify MECS-responsive cells, monitor
the level of polypeptide expression following MECS induction, and
isolate polypeptides directly from animal tissue.
[0034] cDNA libraries enriched for IEG cDNAs can be used to isolate
novel IEG cDNA. Clearly, the isolation of novel IEG cDNAs is
important to further the understanding of brain function. In
addition, isolated nucleic acid derived from such cDNA libraries
can be used to produce polypeptides as well as identify cells that
are responsive to a stimulus such as MECS induction.
[0035] It is important to note that isolated IEG nucleic acid,
cells containing isolated IEG nucleic acid, substantially pure IEG
polypeptides, and anti-IEG polypeptide antibodies can be used to
treat conditions associated with a deficiency in a neuron's ability
to express IEGs in response to a stimulus such as MECS. A condition
associated with a deficiency in a neuron's IEG responsiveness to a
stimulus is any physiological condition characterized as having a
lack of a normal level of responsiveness. For example, when a
deficiency in a neuron's responsiveness to MECS is characterized as
a non- or under-expression of a particular IEG polypeptide by that
neuron, the organism having the condition can be treated with
isolated IEG nucleic acid, cells containing isolated IEG nucleic
acid, or substantially pure IEG polypeptides such that the effect
of the deficiency is minimized. Alternatively, when a deficiency in
a neuron's responsiveness to MECS is characterized as an
over-expression of a particular IEG polypeptide by that neuron, the
organism having the condition can be treated with anti-IEG
polypeptide antibodies or the anti-sense strand of an isolated IEG
nucleic acid such that the effect of the deficiency is
minimized.
[0036] In addition, isolated IEG nucleic acid, cells containing
isolated IEG nucleic acid, substantially pure IEG polypeptides, and
anti-IEG polypeptide antibodies can be used to identify
pharmaceutical compounds that can be used to treat diseases such as
epilepsy, age-dependent memory decline, stroke, and drug addiction.
For example, a compound that modulates IEG nucleic acid expression
or IEG polypeptide activity can be identified by contacting a test
compound with either the IEG nucleic acid or polypeptide, and
determining whether the test compound effects expression or
activity.
[0037] The term "nucleic acid" as used herein encompasses both RNA
and DNA, including cDNA, genomic DNA, and synthetic (e.g.,
chemically synthesized) DNA. The nucleic acid can be
double-stranded or single-stranded. Where single-stranded, the
nucleic acid can be the sense strand or the antisense strand. In
addition, nucleic acid can be circular or linear.
[0038] The term "isolated" as used herein with reference to nucleic
acid refers to a naturally-occurring nucleic acid that is not
immediately contiguous with both of the sequences with which it is
immediately contiguous (one on the 5' end and one on the 3' end) in
the naturally-occurring genome of the organism from which it is
derived. For example, an isolated nucleic acid can be, without
limitation, a recombinant DNA molecule of any length, provided one
of the nucleic acid sequences normally found immediately flanking
that recombinant DNA molecule in a naturally-occurring genome is
removed or absent. Thus, an isolated nucleic acid includes, without
limitation, a recombinant DNA that exists as a separate molecule
(e.g., a cDNA or a genomic DNA fragment produced by PCR or
restriction endonuclease treatment) independent of other sequences
as well as recombinant DNA that is incorporated into a vector, an
autonomously replicating plasmid, a virus (e.g., a retrovirus,
adenovirus, or herpes virus), or into the genomic DNA of a
prokaryote or eukaryote. In addition, an isolated nucleic acid can
include a recombinant DNA molecule that is part of a hybrid or
fusion nucleic acid sequence.
[0039] The term "isolated" also includes any
non-naturally-occurring nucleic acid since non-naturally-occurring
nucleic acid sequences are not found in nature and do not have
immediately contiguous sequences in a naturally-occurring genome.
For example, non-naturally-occurring nucleic acid such as an
engineered nucleic acid is considered to be isolated nucleic acid.
Engineered nucleic acid can be made using common molecular cloning
or chemical nucleic acid synthesis techniques. Isolated
non-naturally-occurring nucleic acid can be independent of other
sequences, or incorporated into a vector, an autonomously
replicating plasmid, a virus (e.g., a retrovirus, adenovirus, or
herpes virus), or the genomic DNA of a prokaryote or eukaryote. In
addition, a non-naturally-occurring nucleic acid can include a
nucleic acid molecule that is part of a hybrid or fusion nucleic
acid sequence.
[0040] It will be apparent to those of skill in the art that a
nucleic acid existing among hundreds to millions of other nucleic
acid molecules within, for example, cDNA or genomic libraries, or
gel slices containing a genomic DNA restriction digest is not to be
considered an isolated nucleic acid.
[0041] Any isolated nucleic acid having a nucleic acid sequence as
set forth in SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14,
15, 16, 17, 18, 19, 20, 22, 23, 24, 25, 26, 28, 29, 31, 33, 34, 35,
37, 39, 40, 41, 42, 43, 44, 45, 46, 47, 49, 50, 51, 52, 53, 54, 55,
56, 57, 58, 59, or 60 is within the scope of the invention. For
convenience, these nucleic acid sequences will be referred to
collectively as the IEG nucleic acid group. In addition, any
isolated nucleic acid having a nucleic acid sequence at least about
60 percent identical (e.g., at least about 65, 70, 75, 80, 85, 90,
95, or 99 percent identical) to a sequence set forth in the IEG
nucleic acid group is within the scope of the invention. For the
purpose of this invention, the percent identity between a sequence
set forth in the IEG nucleic acid group (designated a template
sequence) and any other nucleic acid sequence is calculated as
follows. First, the two nucleic acid sequences are aligned using
the MEGALIGN.RTM. (DNASTAR, Madison, Wis., 1997) sequence alignment
software following the Jotun Heim algorithm with the default
settings. Second, the number of matched positions between the two
aligned nucleic acid sequences is determined. A matched position
refers to a position in which identical bases occur at the same
position as aligned by the MEGALIGN.RTM. sequence alignment
software. Third, the number of matched positions is divided by the
total number of bases in the template sequence, and the resulting
value multiplied by 100 to obtain the percent identity. If the
obtained percent identity is greater than or equal to about 60
percent for a particular nucleic acid sequence, then that
particular nucleic acid sequence is a nucleic acid sequence at
least about 60 percent identical to a sequence set forth in the IEG
nucleic acid group.
[0042] Any isolated nucleic acid having a nucleic acid sequence
that encodes an amino acid sequence at least about 60 percent
identical (e.g., at least about 65, 70, 75, 80, 85, 90, 95, or 99
percent identical) to the sequence set forth in SEQ ID NO:11, 21,
27, 30, 32, 36, 38, 48, 61, or 62 is within the scope of the
invention. For convenience, the amino acid sequences set forth in
SEQ ID NO:11, 21, 27, 30, 32, 36, 38,48,61, and 62 will be referred
to collectively as the IEG amino acid group. For the purpose of
this invention, the percent identity between a sequence set forth
in the IEG amino acid group (designated a template sequence) and
any other amino acid sequence is calculated as follows. First, the
two amino acid sequences are aligned using the MEGALIGN.RTM.
(DNASTAR, Madison, Wis., 1997) sequence alignment software
following the Jotun Heim algorithm with the default settings.
Second, the number of matched positions between the two aligned
amino acid sequences is determined. A matched position refers to a
position in which identical residues occur at the same position as
aligned by the MEGALIGN.RTM. sequence alignment software. Third,
the number of matched positions is divided by the total number of
amino acid residues in the template sequence, and the resulting
value multiplied by 100 to obtain the percent identity. If the
obtained percent identity is greater than or equal to about 60
percent for a particular amino acid sequence, then that particular
amino acid sequence is an amino acid sequence at least about 60
percent identical to a sequence set forth in the IEG amino acid
group.
[0043] Any isolated nucleic acid having a nucleic acid sequence
that encodes an amino acid sequence at least five amino acids in
length also is within the scope of the invention provided the
encoded amino acid sequence has at least three different amino acid
residues, and is identical to a contiguous portion of sequence set
forth in a sequence within the IEG amino acid group.
[0044] Further, any isolated nucleic acid having at least one
adenine base, at least one guanine base, at least one cytosine
base, and at least one thymine or uracil base is within the scope
of the invention provided the isolated nucleic acid is at least
about 12 bases in length (e.g., at least about 14, 15, 16, 17, 18,
19, 20, 25, 30, 40, 50, or 60 bases in length), and hybridizes,
under hybridization conditions, to the sense or antisense strand of
a nucleic acid having a sequence as set forth in the IEG nucleic
acid group. The hybridization conditions can be moderately or
highly stringent hybridization conditions.
[0045] For the purpose of this invention, moderately stringent
hybridization conditions mean the hybridization is performed at
about 42.degree. C. in a hybridization solution containing 25 mM
KPO.sub.4 (pH7.4), 5.times.SSC, 5.times. Denharts solution, 50
.mu.g/ml denatured, sonicated salmon sperm DNA, 50% formamide, 10%
Dextran sulfate, and 1-15 ng/ml probe (>5.times.10.sup.7
cpm/.mu.g), while the washes are performed at about 50.degree. C.
with a wash solution containing 2.times.SSC and 0.1% SDS.
[0046] Highly stringent hybridization conditions mean the
hybridization is performed at about 42.degree. C. in a
hybridization solution containing 25 mM KPO.sub.4 (pH7.4),
5.times.SSC, 5.times. Denharts solution, 50 .mu.g/ml denatured,
sonicated salmon sperm DNA, 50% formamide, 10% Dextran sulfate, and
1-15 ng/ml probe (>5.times.10.sup.7 cpm/.mu.g), while the washes
are performed at about 65.degree. C. with a wash solution
containing 0.2.times.SSC and 0.1% SDS.
[0047] Nucleic acid within the scope of the invention can be
identified and obtained using any method including, without
limitation, common molecular cloning and chemical nucleic acid
synthesis techniques. For example, PCR can be used to obtain a
nucleic acid having a nucleic acid sequence at least about 60
percent identical (e.g., at least about 65, 70, 75, 80, 85, 90, 95,
or, 99 percent identical) to a sequence set forth in the IEG
nucleic acid. group. PCR refers to a procedure or technique in
which target nucleic acid is amplified in a manner similar to that
described in U.S. Pat. No. 4,683,195, and subsequent modifications
of the procedure described therein. Generally, sequence information
from the ends of the region of interest or beyond are used to
design oligonucleotide primers that are identical or similar in
sequence to opposite strands of a potential template to be
amplified. Using PCR, a nucleic acid sequence can be amplified from
RNA or DNA. For example, a nucleic acid sequence can be isolated by
PCR amplification from total cellular RNA, total genomic DNA, and
cDNA as well as from bacteriophage sequences, plasmid sequences,
viral sequences, and the like. When using RNA as a source of
template, reverse transcriptase can be used to synthesize
complimentary DNA strands.
[0048] Nucleic acid within the scope of the invention also can be
obtained by mutagenesis. For example, a nucleic acid sequence set
forth in the IEG nucleic acid group can be mutated using common
molecular cloning techniques (e.g., site-directed mutageneses).
Possible mutations include, without limitation, deletions,
insertions, and base substitutions, as well as combinations of
deletions, insertions, and base substitutions.
[0049] In addition, nucleic acid and amino acid databases (e.g.,
GenBank.RTM.) can be used to identify and obtain a nucleic acid
within the scope of the invention. For example, any nucleic acid
sequence having some homology to a sequence set forth in the IEG
nucleic acid group, or any amino acid sequence having some homology
to a sequence set forth in the IEG amino acid group can be used as
a query to search GenBank.RTM..
[0050] Further, nucleic acid hybridization techniques can be used
to identify and obtain a nucleic acid within the scope of the
invention. Briefly, any nucleic acid having some homology to a
sequence set forth in the IEG nucleic acid group, or fragment
thereof, can be used as a probe to identify a similar nucleic acid
by hybridization under conditions of moderate to high stringency.
Such similar nucleic acid then can be isolated, sequenced, and
analyzed to determine whether they are within the scope of the
invention as described herein.
[0051] Hybridization can be done by Southern or Northern analysis
to identify a DNA or RNA sequence, respectively, that hybridizes to
a probe. The probe can be labeled with a radioisotope such as
.sup.32P, an enzyme, digoxygenin, or by biotinylation. The DNA or
RNA to be analyzed can be electrophoretically separated on an
agarose or polyacrylamide gel, transferred to nitrocellulose,
nylon, or other suitable membrane, and hybridized with the probe
using standard techniques well known in the art such as those
described in sections 7.39-7.52 of Sambrook et al., (1989)
Molecular Cloning, second edition, Cold Spring harbor Laboratory,
Plainview, N.Y. Typically, a probe is at least about 20 nucleotides
in length. For example, a probe corresponding to a 20 nucleotide
sequence set forth in the IEG amino acid group. can be used to
identify a nucleic acid identical to or similar to a nucleic acid
sequence set forth in the IEG nucleic acid group. In addition,
probes longer or shorter than 20 nucleotides can be used.
[0052] Any cell containing an isolated nucleic acid within the
scope of the invention is itself within the scope of the invention.
This includes, without limitation, prokaryotic and eukaryotic
cells. It is noted that cells containing an isolated nucleic acid
of the invention are not required to express the isolated nucleic
acid. In addition, the isolated nucleic acid can be integrated into
the genome of the cell or maintained in an episomal state. In other
words, cells can be stably or transiently transfected with an
isolated nucleic acid of the invention.
[0053] Any method can be used to introduce an isolated nucleic acid
into a cell. In fact, many methods for introducing nucleic acid
into a cell, whether in vivo or in vitro, are well known to those
skilled in the art. For example, calcium phosphate precipitation,
electroporation, heat shock, lipofection, microinjection, and
viral-mediated nucleic acid transfer are common methods that can be
used to introduce nucleic acid into a cell. In addition, naked DNA
can be delivered directly to cells in vivo as describe elsewhere
(U.S. Pat. No. 5,580,859 and U.S. Pat. No. 5,589,466 including
continuations thereof). Further, nucleic acid can be introduced
into cells by generating transgenic animals.
[0054] Transgenic animals can be aquatic animals (such as fish,
sharks, dolphin, and the like), farm animals (such as pigs, goats,
sheep, cows, horses, rabbits, and the like), rodents (such as rats,
guinea pigs, and mice), non-human primates (such as baboon,
monkeys, and chimpanzees), and domestic animals (such as dogs and
cats). Several techniques known in the art can be used to introduce
nucleic acid into animals to produce the founder lines of
transgenic animals. Such techniques include, without limitation,
pronuclear microinjection (U.S. Pat. No. 4,873,191); retrovirus
mediated gene transfer into germ lines (Van der Putten et al.,
Proc. Natl. Acad. Sci., USA, 82:6148-6152 (1985)); gene
transfection into embryonic stem cells (Gossler A et al., Proc Natl
Acad Sci USA 83:9065-9069 (1986)); gene targeting into embryonic
stem cells (Thompson et al., Cell, 56:313-321 (1989)); nuclear
transfer of somatic nuclei (Schnieke A E et al., Science
278:2130-2133 (1997)); and electroporation of embryos.
[0055] For a review of techniques that can be used to generate and
assess transgenic animals, skilled artisans can consult Gordon
(Intl. Rev. Cytol., 115:171-229 (1989)), and may obtain additional
guidance from, for example: Hogan et al., "Manipulating the Mouse
Embryo" Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1986);
Krimpenfort et al., Bio/Technology, 9:844-847 (1991); Palmiter et
al., Cell, 41:343-345 (1985); Kraemer et al., "Genetic Manipulation
of the Early Mammalian Embryo" Cold Spring Harbor Press, Cold
Spring Harbor, N.Y.(1985); Hammer et al., Nature, 315:680-683
(1985); Purscel et al., Science, 244:1281-1288 (1986); Wagner et
al., U.S. Pat. No. 5,175,385; and Krimpenfort et al., U.S. Pat. No.
5,175,384.
[0056] Any method can be used to identify cells that contain an
isolated nucleic acid within the scope of the invention. For
example, PCR and nucleic acid hybridization techniques such as
Northern and Southern analysis can be used. In some cases,
immunohistochemistry and biochemical techniques can be used to
determine if a cell contains a particular nucleic acid by detecting
the expression of a polypeptide encoded by that particular nucleic
acid. For example, detection of polypeptide X-immunoreactivity
after introduction of an isolated nucleic acid containing a cDNA
that encodes polypeptide X into a cell that does not normally
express polypeptide X can indicate that that cell not only contains
the introduced nucleic acid but also expresses the encoded
polypeptide X from that introduced nucleic acid. In this case, the
detection of any enzymatic activities of polypeptide X also can
indicate that that cell contains the introduced nucleic acid and
expresses the encoded polypeptide X from that introduced nucleic
acid.
[0057] In addition, any method can be used to force a cell to
express an encoded amino acid sequence from a nucleic acid. Such
methods are well known to those skilled in the art, and include,
without limitation, constructing a nucleic acid such that a
regulatory element drives the expression of a nucleic acid sequence
that encodes a polypeptide. Typically, regulatory elements are DNA
sequences that regulate the expression of other DNA sequences at
the level of transcription. Such regulatory elements include,
without limitation, promoters, enhancers, and the like. Further,
any methods can be used to identifying cells that express an amino
acid sequence from a nucleic acid. Such methods are well known to
those skilled in the art, and include, without limitation,
immunocytochemistry, Northern analysis, and RT-PCR.
[0058] The term "substantially pure" as used herein with reference
to a polypeptide means the polypeptide is substantially free of
other polypeptides, lipids, carbohydrates, and nucleic acid with
which it is naturally associated. Thus, a substantially pure
polypeptide is any polypeptide that is removed from its natural
environment and is at least 60 percent free, preferably 75 percent
free, and most preferably 90 percent free from other components
with which it is naturally associated. Typically, a substantially
pure polypeptide will yield a single major band on a non-reducing
polyacrylamide gel.
[0059] Any substantially pure polypeptide having an amino acid
sequence encoded by a nucleic acid within the scope of the
invention is itself within the scope of the invention. In addition,
any substantially pure polypeptide having an amino acid sequence at
least about 60 percent (e.g., at least about 65, 70, 75, 80, 85,
90, 95, or 99 percent) identical to a sequence set forth in the IEG
amino acid group is within the scope of the invention. The percent
identity between particular amino acid sequences is determined as
described herein.
[0060] Any method can be used to obtain a substantially pure
polypeptide. For example, one skilled in the art can use common
polypeptide purification techniques such as affinity chromotography
and HPLC as well as polypeptide synthesis techniques. In addition,
any material can be used as a source to obtain a substantially pure
polypeptide. For example, tissue from wild-type or transgenic
animals can be used as a source material. In addition, tissue
culture cells engineered to overexpress a particular polypeptide of
interest can be used to obtain substantially pure polypeptide.
Further, a polypeptide within the scope of the invention can be
"engineered" to contain an amino acid sequence that allows the
polypeptide to be captured onto an affinity matrix. For example, a
tag such as c-myc, hemagglutinin, polyhistidine, or Flag.RTM. tag
(Kodak) can be used to aid polypeptide purification. Such tags can
be inserted anywhere within the polypeptide including at either the
carboxyl or amino termini. Other fusions that could be useful
include enzymes that aid in the detection of the polypeptide, such
as alkaline phosphatase.
[0061] The term "antibody" as used herein refers to intact
antibodies as well as antibody fragments that retain some ability
to selectively bind an epitope. Such fragments include, without
limitation, Fab, F(ab')2, and Fv antibody fragments. The term
"epitope" refers to an antigenic determinant on an antigen to which
the paratope of an antibody binds. Epitopic determinants usually
consist of chemically active surface groupings of molecules (e.g.,
amino acid or sugar residues) and usually have specific three
dimensional structural characteristics as well as specific charge
characteristics.
[0062] Any antibody having specific binding affinity for an amino
acid sequence encoded by a nucleic acid within the scope of the
invention is itself within the scope of the invention. Thus, any
monoclonal or polyclonal antibody having specific binding affinity
for an amino acid sequence set forth in the IEG amino acid group is
within the scope of the invention. Such antibodies can be used in
immunoassays in liquid phase or bound to a solid phase. For
example, the antibodies of the invention can be used in competitive
and non-competitive immunoassays in either a direct or indirect
format. Examples of such immunoassays include the radioimmunoassay
(RIA) and the sandwich (immunometric) assay.
[0063] Antibodies within the scope of the invention can be prepared
using any method. For example, any substantially pure polypeptide
provided herein, or fragment thereof, can be used as an immunogen
to elicit an immune response in an animal such that specific
antibodies are produced. Thus, an intact full-length polypeptide or
fragments containing small peptides can be used as an immunizing
antigen. In addition, the immunogen used to immunize an animal can
be chemically synthesized or derived from translated cDNA. Further,
the immunogen can be conjugated to a carrier polypeptide, if
desired. Commonly used carriers that are chemically coupled to an
immunizing polypeptide include, without limitation, keyhole limpet
hemocyanin (KLH), thyroglobulin, bovine serum albumin (BSA), and
tetanus toxoid.
[0064] The preparation of polyclonal antibodies is well-known to
those skilled in the art. See, e.g., Green et al., Production of
Polyclonal Antisera, in IMMUOCHEMICAL PROTOCOLS (Manson, ed.),
pages 1-5 (Humana Press 1992) and Coligan et al., Production of
Polyclonal Antisera in Rabbits, Rats, Mice and Hamsters, in CURRENT
PROTOCOLS IN IMMUNOLOGY, section 2.4.1 (1992). In addition, those
of skill in the art will know of various techniques common in the
immunology arts for purification and concentration of polyclonal
antibodies, as well as monoclonal antibodies (Coligan, et al., Unit
9, Current Protocols in Immunology, Wiley Interscience, 1994).
[0065] The preparation of monoclonal antibodies also is well-known
to those skilled in the art. See, e.g., Kohler & Milstein,
Nature 256:495 (1975); Coligan et al., sections 2.5.1-2.6.7; and
Harlow et al., ANTIBODIES: A LABORATORY MANUAL, page 726 (Cold
Spring Harbor Pub. 1988). Briefly, monoclonal antibodies can be
obtained by injecting mice with a composition comprising an
antigen, verifying the presence of antibody production by analyzing
a serum sample, removing the spleen to obtain B lymphocytes, fusing
the B lymphocytes with myeloma cells to produce hybridomas, cloning
the hybridomas, selecting positive clones that produce antibodies
to the antigen, and isolating the antibodies from the hybridoma
cultures. Monoclonal antibodies can be isolated and purified from
hybridoma cultures by a variety of well-established techniques.
Such isolation techniques include affinity chromatography with
Protein-A Sepharose, size-exclusion chromatography, and
ion-exchange chromatography. See, e.g., Coligan et al. , sections
2.7.1-2.7.12 and sections 2.9.1-2.9.3; Barnes et al., Purification
of Immunoglobulin G (IgG), in METHODS IN MOLECULAR BIOLOGY, VOL.
10, pages 79-104 (Humana Press 1992).
[0066] In addition, methods of in vitro and in vivo multiplication
of monoclonal antibodies is well-known to those skilled in the art.
Multiplication in vitro can be carried out in suitable culture
media such as Dulbecco's Modified Eagle Medium or RPMI 1640 medium,
optionally replenished by mammalian serum such as fetal calf serum,
or trace elements and growth-sustaining supplements such as normal
mouse peritoneal exudate cells, spleen cells, and bone marrow
macrophages. Production in vitro provides relatively pure antibody
preparations and allows scale-up to yield large amounts of the
desired antibodies. Large scale hybridoma cultivation can be
carried out by homogenous suspension culture in an airlift reactor,
in a continuous stirrer reactor, or in immobilized or entrapped
cell culture. Multiplication in vivo may be carried out by
injecting cell clones into mammals histocompatible with the parent
cells. (e.g., osyngeneic mice) to cause growth of
antibody-producing tumors. Optionally, the animals are primed with
a hydrocarbon, especially oils such as pristane
(tetramethylpentadecane) prior to injection. After one to three
weeks, the desired monoclonal antibody is recovered from the body
fluid of the animal.
[0067] The antibodies within the scope of the invention also can be
made using non-human primates. General techniques for raising
therapeutically useful antibodies in baboons can be found, for
example, in Goldenberg et al., International Patent Publication WO
91/11465 (1991) and Losman et al., Int. J. Cancer 46:310
(1990).
[0068] Alternatively, the antibodies can be "humanized" monoclonal
antibodies. Humanized monoclonal antibodies are produced by
transferring mouse complementarity determining regions (CDRs) from
heavy and light variable chains of the mouse immunoglobulin into a
human variable domain, and then substituting human residues in the
framework regions of the murine counterparts. The use of antibody
components derived from humanized monoclonal antibodies obviates
potential problems associated with the immunogenicity of murine
constant regions when treating humans. General techniques for
cloning murine immunoglobulin variable domains are described, for
example, by Orlandi et al., Proc. Nat'l. Acad. Sci. USA 86:3833
(1989). Techniques for producing humanized monoclonal antibodies
are described, for example, by Jones et al., Nature 321:522 (1986);
Riechmann et al., Nature 332:323 (1988); Verhoeyen et al., Science
239:1534 (1988); Carter et al., Proc. Nat'l. Acad. Sci. USA 89:4285
(1992); Sandhu, Crit. Rev. Biotech. 12:437 (1992); and Singer et
al., J. Immunol. 150:2844 (1993).
[0069] Antibodies of the present invention also may be derived from
human antibody fragments isolated from a combinatorial
immunoglobulin library. See, for example, Barbas et al., METHODS: A
COMPANION TO METHODS IN ENZYMOLOGY, VOL. 2, page 119 (1991) and
Winter et al., Ann. Rev. Immunol. 12: 433 (1994). Cloning and
expression vectors that are useful for producing a human
immunoglobulin phage library can be obtained, for example, from
STRATAGENE Cloning Systems (La Jolla, Calif.).
[0070] In addition, antibodies of the present invention may be
derived from a human monoclonal antibody. Such antibodies are
obtained from transgenic mice that have been "engineered" to
produce specific human antibodies in response to antigenic
challenge. In this technique, elements of the human heavy and light
chain loci are introduced into strains of mice derived from
embryonic stem cell lines that contain targeted disruptions of the
endogenous heavy and light chain loci. The transgenic mice can
synthesize human antibodies specific for human antigens and can be
used to produce human antibody-secreting hybridomas. Methods for
obtaining human antibodies from transgenic mice are described by
Green et al., Nature Genet. 7:13 (1994); Lonberg et al., Nature
368:856 (1994); and Taylor et al., Int. Immunol. 6:579 (1994).
[0071] Antibody fragments of the present invention can be prepared
by proteolytic hydrolysis of an intact antibody or by the
expression of a nucleic acid encoding the fragment. Antibody
fragments can be obtained by pepsin or papain digestion of intact
antibodies by conventional methods. For example, antibody fragments
can be produced by enzymatic cleavage of antibodies with pepsin to
provide a 5S fragment denoted F(ab').sub.2. This fragment can be
further cleaved using a thiol reducing agent, and optionally a
blocking group for the sulfhydryl groups resulting from cleavage of
disulfide linkages, to produce 3.5S Fab' monovalent fragments.
Alternatively, an enzymatic cleavage using pepsin produces two
monovalent Fab' fragments and an Fc fragment directly. These
methods are described, for example, by Goldenberg (U.S. Pat. Nos.
4,036,945 and 4,331,647). See also Nisonhoff et al., Arch. Biochem.
Biophys. 89:230 (1960); Porter, Biochem. J. 73:119 (1959); Edelman
et al., METHODS IN ENZYMOLOGY, VOL. 1, page 422 (Academic Press
1967); and Coligan et al. at sections 2.8.1-2.8.10 and
2.10.1-2.10.4.
[0072] Other methods of cleaving antibodies, such as separation of
heavy chains to form monovalent light-heavy chain fragments,
further cleavage of fragments, or other enzymatic, chemical, or
genetic techniques may also be used provided the fragments retain
some ability to selectively bind its epitope.
[0073] For example, Fv fragments comprise an association of V.sub.H
and V.sub.L chains. This association may be noncovalent, as
described in Inbar et al., Proc. Nat'l. Acad. Sci. USA 69:2659
(1972). Alternatively, the variable chains can be linked by an
intermolecular disulfide bond or cross-linked by chemicals such as
glutaraldehyde. See, e.g., Sandhu, supra. Preferably, the Fv
fragments comprise V.sub.H and V.sub.L chains connected by a
peptide linker. These single-chain antigen binding polypeptides
(sFv) are prepared by constructing a nucleic acid construct
encoding the V.sub.H and V.sub.L domains connected by an
oligonucleotide. This nucleic acid construct is inserted into an
expression vector, which is subsequently introduced into a host
cell such as E. coli. The recombinant host cells synthesize a
single polypeptide chain with a linker peptide bridging the two V
domains. Methods for producing sFvs are described, for example, by
Whitlow et al., METHODS: A COMPANION TO METHODS IN ENZYMOLOGY, VOL.
2, page 97 (1991); Bird et al., Science 242:423-426 (1988); Ladner
et al., U.S. Pat. No. 4,946,778; Pack et al., Bio/Technology
11:1271-77 (1993); and Sandhu, supra.
[0074] Another form of an antibody fragment is a peptide coding for
a single CDR. CDR peptides ("minimal recognition units") can be
obtained by constructing nucleic acid constructs that encode the
CDR of an antibody of interest. Such constructs are prepared, for
example, by using PCR to synthesize the variable region from RNA of
antibody-producing cells. See, e.g., Larrick et al., METHODS: A
COMPANION TO METHODS IN ENZYMOLOGY, VOL. 2, page 106 (1991).
[0075] It is also possible to use anti-idiotype technology to
produce monoclonal antibodies that mimic an epitope. For example,
an anti-idiotypic monoclonal antibody made to a first monoclonal
antibody will have a binding domain in the hypervariable region
that is the "image" of the epitope bound by the first monoclonal
antibody. Such anti-idiotypic monoclonal antibodies can be used to
inhibit the activity of the polypeptide containing the original
epitope.
[0076] The invention also provides cDNA libraries enriched for
IEGs. As described herein, such cDNA libraries contain an increased
frequency of cDNAs derived from IEGs. Specifically, about 15
percent (e.g., about 20 or 25 percent) of the cDNA clones within
the cDNA libraries provided herein are derived from IEGs.
[0077] A cDNA library within the scope of the invention can be
prepared from any tissue containing cells that express an IEG
(e.g., hippocampus tissue). Again, an IEG is a gene whose
expression is rapidly increased immediately following a stimulus.
The stimulus can be electrical or chemical in nature. For example,
cells can be treated with electric shock or chemicals such as
kainate. Briefly, cDNA libraries are prepared from the hippocampus
of control animals (e.g., rats) as well as from animals that
receive a stimulus (e.g., multiple MECS) using, for example, a
phage vector lambda ZAP II (Stratagene). A subtracted library is
then prepared using in vitro mRNA prepared from a control library
and subsequent solution phase hybridization with cDNA prepared from
a stimulated library. The control in vitro mRNA can be tagged with
biotin to permit its removal from solution using avidin beads
(Lanahan et al., Mol. Cell. Biol. 12:3919-3929 (1992)). cDNA that
remains after removal of mRNA/cDNA hybrids can be recloned into,
for example, a lambda ZAPII phage vector. Several rounds of
subtraction (e.g., two, three, four, or five rounds) can be used to
increase the frequency of IEGs. The subtracted library then can be
plated and duplicate phage lifts screened with a radiolabeled cDNA
probe. Any probe can be used provided it contains at least one
nucleic acid sequence derived from an IEG. For example, a probe can
be prepared from mRNA obtained from the hippocampus of a stimulated
animal. In addition, the mRNA used to make a probe can be subjected
to subtractive hybridization such that IEG sequences are enriched.
In general, conventional cDNA libraries contain IEGs at a frequency
of<1:30,000 cDNAs. For the cDNA libraries enriched for IEGs,
however, about 1 in 5 genes can be induced by a stimulus such as
MECS. This represents an about 1000 to 10,000 fold enrichment in
brain IEGs.
[0078] An animal (e.g., human) having a deficiency in a neuron's
IEG responsiveness to a stimulus (e.g., a stimulus that influences
learning or memory) can be treated using the methods and materials
described herein. A stimulus that influences learning or memory can
be a multiple MECS treatment. A deficiency in a neuron's IEG
responsiveness to a stimulus means the level of IEG responsiveness
is not normal. Such deficiencies can be identified by stimulating a
sample of cells and measuring the levels of IEG expression. If the
levels are not similar to the levels normally observed in a similar
tissue sample, then there is a deficiency. It is noted that
increased IEG expression as well as decreased IEG expression can be
classified as a deficiency provided the levels are not normal.
[0079] A deficiency in a neuron's IEG responsiveness to a stimulus
can be treated by administering a nucleic acid of the invention to
the animal such that the effect of the deficiency is minimized. The
administration can be an in vivo, in vitro, or ex vivo
administration as described herein. For example, an in vivo
administration can involve administering a viral vector to the
hippocampal region of an animal, while an ex vivo administration
can involve extracting cells from an animal, transfecting the cells
with the nucleic acid in tissue culture, and then introducing the
transfected cells back into the same animal.
[0080] In addition, a deficiency in a neuron's IEG responsiveness
to a stimulus can be treated by administering a therapeutically
effective amount cells containing isolated IEG nucleic acid,
substantially pure IEG polypeptides, anti-IEG polypeptide
antibodies, or combinations thereof. A therapeutically effective
amount is any amount that minimizes the effect of the deficiency
while not causing significant toxicity to the animal. Such an
amount can be determined by assessing the clinical symptoms
associated with the deficiency before and after administering a
fixed amount of cells, polypeptides, or antibodies. In addition,
the effective amount administered to an animal can be adjusted
according to the animal's response and desired outcomes.
Significant toxicity can vary for each particular patient and
depends on multiple factors including, without limitation, the
patient's physical and mental state, age, and tolerance to pain.
The cells, polypeptides, or antibodies can be administered to any
part of the animal's body including, without limitation, brain,
spinal cord, blood stream, muscle tissue, skin, peritoneal cavity,
and the like. Thus, these therapeutic agents can be administered by
injection (e.g., intravenous, intraperitoneal, intramuscular,
subcutaneous, intracavity, or transdermal injection) or by gradual
perfusion over time.
[0081] Preparations for administration can include sterile aqueous
or non-aqueous solutions, suspensions, and emulsions. Examples of
non-aqueous solvents are propylene glycol, polyethylene glycol,
vegetable oils such as olive oil, and injectable organic esters
such as ethyl oleate. Aqueous carriers include water,
alcoholic/aqueous solutions, emulsions or suspensions, including
saline and buffered media. Other vehicles for adminstration include
sodium chloride solution, Ringer's dextrose, dextrose and sodium
chloride, lactated Ringer's intravenous vehicles containing fluid
and nutrient replenishers, electrolyte replenishers (such as those
based on Ringer's dextrose), and the like. Preservatives and other
additives may also be present such as, for example, antimicrobials,
anti-oxidants, chelating agents, and inert gases and the like.
[0082] Further, a deficiency in a neuron's IEG responsiveness to a
stimulus can be treated by administering a therapeutically
effective amount of a compound that directly interferes with the
translation of IEG nucleic acid. For example, antisense nucleic
acid or ribozymes could be used to bind to IEG mRNA or to cleave
it. Antisense RNA or DNA molecules bind specifically with a
targeted RNA message, interrupting the expression of the mRNA
product. The antisense binds to the messenger RNA forming a double
stranded molecule that cannot be translated by the cell. Typically,
an antisense oligonucleotides is about 15-25 nucleotides in length.
In addition, chemically reactive groups, such as iron-linked
ethylenediaminetetraacetic acid (EDTA-Fe), can be attached to an
antisense oligonucleotide, causing cleavage of the mRNA at the site
of hybridization. These and other uses of antisense methods to
inhibit the translation of nucleic acid are well known in the art
(Marcus-Sakura, Anal. Biochem., 172:289 (1988)).
[0083] An oligonucleotide also can be used to stall transcription
winding around double-helical DNA and forming a three-strand helix
(Maher, et al., Antisense Res. and Dev., 1:227 (1991) and Helene,
Anticancer Drug Design, 6:569 (1991)).
[0084] Ribozymes are RNA molecules possessing the ability to
specifically cleave other single-stranded RNA in a manner analogous
to DNA restriction endonucleases. By modifying nucleic acid
sequences that encode ribozymes, it is possible to engineer
molecules that recognize specific nucleotide sequences in an RNA
molecule and cleave it (Cech, J. Amer. Med. Assn., 260:3030
(1988)). There are two basic types of ribozymes namely,
tetrahymena-type (Hasselhoff, Nature, 334:585 (1988)) and
"hammerhead"-type. Tetrahymena-type ribozymes recognize sequences
that are four bases in length, while "hammerhead"-type ribozymes
recognize sequences 11-18 bases in length. The longer the
recognition sequence, the greater the likelihood that the sequence
will occur exclusively in the target mRNA species. Consequently,
"hammerhead"-type ribozymes are preferable to tetrahymena-type
ribozymes for inactivating a specific mRNA species. In addition,
18-based recognition sequences are preferable to shorter
recognition sequences. These and other uses of antisense methods to
inhibit the in vivo translation of nucleic acid are well known in
the art (DeMesmaeker et al., Curr. Opin. Struct. Biol. 5:343-355
(1995); Gewirtz et al., Proc. Nat'l. Acad. Sci. U.S.A.,
93:3161-3163 (1996); and Stein, Chem. Biol. 3:319-323 (1996)).
[0085] Delivery of nucleic acid, antisense, triplex agents, and
ribozymes can be achieved using a recombinant expression vector
such as a chimeric virus or a colloidal dispersion system. Various
viral vectors that can be utilized for gene therapy include
adenoviruses, herpesviruses, vaccinia viruses, and retroviruses. A
retroviral vector can be a derivative of a murine or avian
retrovirus. Examples of retroviral vectors in which a single
foreign gene can be inserted include, but are not limited to:
Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus
(HaMuSV), murine mammary tumor virus (MuMTV), and Rous Sarcoma
Virus (RSV). A number of additional retroviral vectors can
incorporate multiple genes. All of these vectors can transfer or
incorporate a gene for a selectable marker so that transduced cells
can be identified and generated. In addition, a nucleic acid
sequence of interest along with another nucleic acid sequence that
encodes a ligand for a receptor on a specific target cell can be
inserted into a viral vector to produce a vector that is target
specific. For example, retroviral vectors can be made target
specific by inserting a nucleic acid sequence that encodes an
antibody that binds a specific target antigen. Those of skill in
the art can readily ascertain without undue experimentation
specific nucleic acid sequences that can be inserted into a
retroviral genome to allow target specific delivery of the
retroviral vector containing the nucleic acid of the invention.
[0086] A colloidal dispersion system can be used to target the
delivery of the nucleic acid of the invention. Colloidal dispersion
systems include macromolecule complexes, nanocapsules,
microspheres, beads, and lipid-based systems including oil-in-water
emulsions, micelles, mixed micelles, and liposomes. Liposomes are
artificial membrane vesicles that are useful as delivery vehicles
in vitro and in vivo. It has been shown that large unilamellar
vesicles (LUV) that range in size from 0.2-4.0 .mu.m can
encapsulate a substantial percentage of an aqueous buffer
containing large macromolecules. Thus, nucleic acid, intact
virions, polypeptides, and antibodies can be encapsulated within
the aqueous interior and be delivered to cells in a biologically
active form (Fraley et al., Trends Biochem. Sci., 6:77 (1981)). In
addition to mammalian cells, liposomes have been used to deliver
nucleic acid to plants, yeast, and bacteria. In order for a
liposome to be an efficient nucleic acid transfer vehicle, the
following characteristics should be present: (1) encapsulation of
the nucleic acid of interest at high efficiency while not
compromising its biological activity; (2) preferential and
substantial binding to a target cell in comparison to non-target
cells; (3) delivery of the aqueous contents of the vesicle to the
target cell cytoplasm at high efficiency; and (4) accurate and
effective expression of the nucleic acid (Mannino et al.,
Biotechniques, 6:682 (1988).
[0087] The composition of a liposome is usually a combination of
phospholipids, particularly high-phase-transition-temperature
phospholipids, usually in combination with steroids, especially
cholesterol. Other phospholipids or other lipids also can be used.
The physical characteristics of liposomes depend on pH, ionic
strength, and the presence of divalent cations.
[0088] Examples of lipids useful in liposome production include
phosphatidyl compounds, such as phosphatidylglycerol,
phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine,
sphingolipids, cerebrosides, and gangliosides. Particularly useful
are diacylphosphatidylglycerols, where the lipid moiety contains
from 14-18 carbon atoms, particularly from 16-18 carbon atoms, and
is saturated. Illustrative phospholipids include egg
phosphatidylcholine, dipalmitoylphosphatidylcholine, and
distearoylphosphatidylcholine.
[0089] The surface of the targeted delivery system may be modified
in a variety of ways. In the case of a liposomal targeted delivery
system, lipid groups can be incorporated into the lipid bilayer of
the liposome in order to maintain the targeting ligand in stable
association with the liposomal bilayer. Various linking groups can
be used for joining the lipid chains to the targeting ligand. In
general, the compounds bound to the surface of the targeted
delivery system will be ligands and receptors that allow the
targeted delivery system to find and "home in" on the desired
cells. A ligand may be any compound of interest that will bind to
another compound, such as a receptor or antibody.
[0090] Compounds that modulate IEG expression can be identified by
contacting a test compound with an IEG nucleic acid, and
determining whether the test compound effects expression. Likewise,
compounds that modulate IEG polypeptide activity can be identified
by contacting a test compound with an IEG polypeptide, and
determining whether the test compound effects polypeptide activity.
Contacting includes in solution and in solid phase, or in a cell.
Any type of compound can be used as a test compound including,
without limitation, peptides, peptidomimetics, polypeptides,
chemical compounds, and biologic agents. In addition, the test
compound can be a combinatorial library for screening a plurality
of compounds. Compounds identified using the method of the
invention can be further evaluated, detected, cloned, sequenced,
and the like, either in solution or after binding to a solid
support, by any method usually applied to the detection of a
specific DNA sequence such as PCR, oligomer restriction (Saiki, et
al., Bio/Technology, 3:1008-1012, 1985), allele-specific
oligonucleotide (ASO) probe analysis (Conner, et al., Proc. Nat'l.
Acad. Sci. USA, 80:278 (1983), oligonucleotide ligation assays
(OLAs; Landegren, et al., Science, 241:1077 (1988), and the
like.
[0091] The invention will be further described in the following
examples, which do not limit the scope of the invention described
in the claims.
EXAMPLES
Example 1
Construction of Subtracted cDNA Libraries
[0092] The mRNA used to prepare the cDNA libraries was obtained
from the hippocampus of adult rats (male or female). Briefly, the
hippocampus was dissected from naive or stimulated rats, and
rapidly frozen in liquid nitrogen. The stimulation protocol used to
stimulate the rats was as follows. Rats were injected
intraperitoneally with 50 mg of the protein synthesis inhibitor
cycloheximide (50 mg/ml stock in 50% ethanol) per kilogram of body
weight 15 minutes prior to initiating repetitions of maximal
electroconvulsive seizure (MECS). MECS was induced by passage of a
constant current signal by means of an ECT unit (Ugo, Basil). The
current signal lasted one second with a frequency of 100 Hz. Each
pulse lasted 0.5 milliseconds, and the current was 90 milliamperes.
This stimulus caused brief loss of consciousness and a tonic-clonic
seizure lasting 30 seconds to one minute. MECS was administered
about every 15 minutes for a total of 13 administrations over the
course of 2.5 to 3 hours. Thirty (30) minutes after the last MECS,
the rats were sacrificed by decapitation.
[0093] To collect total RNA, the tissue was homogenized in 4M
guanidinium thiocynate using a polytron and then centrifuged
through a CsCl cushion. To isolate polyA.sup.+ RNA, the resulting
total RNA was chromatographed on oligo(dT) columns using a
commercial oligo(dT) resin and purification protocol (Fastback,
Invitrogen). About 50 naive (control) and 50 stimulated rats were
used to generate the polyA.sup.+ mRNA needed to make the cDNA
libraries and perform the Northern blot analysis.
[0094] A nonsubtracted cDNA library was made using polyA.sup.+ RNA
isolated from rats subjected to MECS. Briefly, cDNA was synthesized
and cloned directionally into the Lambda ZAP vector yielding a
library containing 3.6.times.10.sup.6 recombinants. This library
was designated the 3 hr MECS/CHX library. Differential screening of
the 3 hr MECS/CHX library with control and stimulated rat
hippocampal cDNA probes yielded several novel IEGs. Analysis of
these IEGs revealed that they were relatively abundant.
[0095] The 3 hr MECS/CHX library was used as starting material for
preparing a subtracted cDNA library highly enriched for IEGs. A
subtracted cDNA library highly enriched for IEGs can allow for the
detection of lower abundance novel IEGs. To make a subtracted cDNA
library, DNA template was prepared from the 3 hr MECS/CHX library
as follows.
[0096] The 3 hr MECS/CHX library was amplified and plated on 15 cm
NZCYM agarose plates at a density of about 50,000 phage/plate. A
total of 1.85.times.10.sup.6 phage were plated on a total of 37
plates. The plates were overlaid with Suspension Media (SM) and the
phage particles eluted by swirling at 4.degree. C. overnight. The
lysate was collected, and chloroform added to a final concentration
of 5%. The lysate was clarified by centrifugation, and the phage
containing supernatant collected and stored at 4.degree. C. A 300
ml aliquot of the lysate was treated with RNaseA (final
concentration of 1 .mu.g/.mu.l) and DNase I (final concentration of
1 .mu.g/.mu.l) for three hours at 37.degree. C. Polyethylene glycol
(PEG 6000) was added to a concentration of 10%, and NaCl added to a
concentration of 1 M. After mixing well, the lysate was stored at
4.degree. C. overnight to allow phage particles to precipitate.
Phage particles were pelleted by centrifugation, resuspended in 20
ml of SM, and stored at 4.degree. C. Phage particles were lysed by
adding EDTA to a concentration of 10 mM and SDS to a concentration
of 0.2% followed by a 20 minute incubation at 68.degree. C.
Polypeptides were removed by two extractions with
phenol/chloroform/isoamyl alcohol (50:48:2) followed by two
extractions with chloroform/isoamyl alcohol (24:1). The phage DNA
contained within 40 ml of lysate was precipitated by adding
{fraction (1/10)}th volume of 3M NaOAc (pH 5.2) followed by the
addition of 2 volumes of 100% ethanol. After mixing, the solution
was incubated at -20.degree. C. overnight. DNA was pelleted by
centrifugation, resuspended in 10 mM Tris, 1 mM EDTA pH 7.5 (TE),
and reprecipitated overnight. After this second precipitation, the
DNA was pelleted by centrifugation and resuspended in 12 ml of 10
mM Tris (pH 7.5), 5 mM EDTA, 300 mM NaCl. To remove residual RNA,
RNase A (final concentration of 50 .mu.g/ml) was added followed by
incubation at 37.degree. C. for 1 hour. To remove RNase A, SDS
(final concentration of 0.5%) and then Proteinase K (final
concentration of 50 .mu.g/ml) was added followed incubation at
37.degree. C. for 1 hour. The DNA lysate was extracted twice with
phenol/chloroform/isoamyl alcohol (50:48:2) followed by one
extraction with chloroform/isoamyl alcohol (24:1). After this
extraction, the DNA lysate was dialyzed against 12 liters of TE for
2 days at 4.degree. C. The 300 ml aliquot of phage lysate yielded
7254 .mu.g of phage DNA. This phage DNA was then used to prepare in
vitro polyA.sup.+ RNA (cRNA).
[0097] To prepare in vitro cRNA, the phage DNA template was
linearized at the 3' end of the cDNA insert using the restriction
enzyme XhoI. Briefly, 1 mg of phage DNA was digested with 1000 U of
XhoI for three hours at 37.degree. C. After the three hour
incubation, an additional 1000 U of XhoI was added and the
37.degree. C. incubation continued an additional three hours. XhoI
was removed by adding SDS to 0.5% and Proteinase K to 50 .mu.g/ml
followed by incubation at 37.degree. C. for one hour. Polypeptides
were removed by three extractions with phenol/chloroform/isoamyl
alcohol (50:48:2) followed by one extraction with
chloroform/isoamyl alcohol (24:1). The DNA was precipitated with
{fraction (1/10)}th volume 3M NaOAc (pH 5.2) and 2 volumes 100%
ethanol. The DNA was pelleted by centrifugation and resuspended in
500 .mu.l TE (1.58 mg/ml final DNA concentration).
[0098] This linearized DNA was used as template to prepare in vitro
cRNA from the sense strand of the cDNA inserts. This cRNA is
representative of the initial in vivo population of RNA in the
MECS/cycloheximide treated rat hippocampus. Forty (40) .mu.g of DNA
template was incubated with 40 mM Tris (pH 7.5), 6 mM MgCl.sub.2, 2
mM spermidine, 10 mM NaCl, 10 mM DTT, 1 U/.mu.l RNasin, 500 .mu.M
ATP, 500 .mu.M CTP, 500 .mu.M GTP, 500 .mu.M UTP, and 2 U/.mu.l T3
RNA polymerase in a final volume of 300 .mu.l for two hours at
40.degree. C. After two hours, an additional 2 U/.mu.l of T3 RNA
polymerase was added, and the reaction incubated for an additional
two hours at 37.degree. C. for a total time of four hours. The DNA
template was removed by adding DNaseI (2 U/.mu.g of template) and
incubating the mixture at 37.degree. C. for an hour. Polypeptides
were removed by two extractions with phenol/chloroform/isoamyl
alcohol (50:48:2) followed by one extraction with
chloroform/isoamyl alcohol (24:1). The cRNA was precipitated at
20.degree. C. with one half volume 7.5 M NH.sub.4OAc and 2 volumes
100% ethanol. The cRNA was pelleted and resuspended in TE. The cRNA
was chromatographed on sephadex G-50 columns (NICK columns;
Pharmacia) to remove free nucleotides and the concentration of cRNA
determined by UV absorbance at 260 A. Thirty (30) .mu.g of DNA
template yielded 68.6 .mu.g of cRNA. The cRNA was either stored
frozen at -20.degree. C. or precipitated with {fraction (1/10)}th
volume 2 M KOAc (pH 5) and 2 volumes 100% ethanol. The 68.6 .mu.g
of cRNA was further purified using oligo(dT) column chromotography
to select polyA.sup.+ cRNA. The cRNA was bound to oligo(dT) under
high salt conditions, rinsed with low salt conditions, and eluted
with TE (pH 7.5). This eluted cRNA was again passed over an
oligo(dT) column under high salt conditions, rinsed with low salt
conditions, and the polyA.sup.+ cRNA eluted with TE (pH 7.5). The
two passes on oligo(dT) cellulose yielded 34.2 .mu.g of polyA.sup.+
cRNA. This polyA.sup.+ cRNA was then used as template for synthesis
of first strand cDNA that was then subtracted against control brain
and liver polyA.sup.+ RNA.
[0099] Two cDNA synthesis reactions were performed to prepare first
strand cDNA from the polyA.sup.+ cRNA. One involved using 2 .mu.g
of cRNA with a small amount of .sup.32P-dCTP to allow for the
analysis of subtraction efficiency, and the other involved using 5
.mu.g of cRNA with no radioactive dNTPs. The radioactive cDNA
synthesis reaction was as follows. First, 2 .mu.l cRNA (1
.mu.g/.mu.l in TE), 1 .mu.l Xho(dT) primer (1.4 .mu.g/.mu.l), and 8
.mu.l water was combined, and the mixture was incubated at
70.degree. C. for ten minutes, quickly spun, and placed on ice.
Second, 1 .mu.l RNasin (40 U/.mu.l), 5 .mu.l 5.times. Reaction
Buffer (BRL), 2.5 .mu.l 0.1M DTT, 1.5 .mu.l dNTP mix, and 2 .mu.l
.sup.32P dCTP (3000 Ci/mmole) was added, and the mixture was
incubated at room temperature for ten minutes. The dNTP mix
contained 10 mM of each dATP, dGTP, and dTTP as well as 5 mM of
methyl dCTP. After incubation, 2 .mu.l of Superscript/MMLV RT mix
(1:1) was added, and the mixture (25 .mu.l total volume) was
incubated at room temperature for five minutes followed by a 90
minute incubation at 40.degree. C. The nonradioactive cDNA
synthesis reaction was as follows. First, 5 .mu.l cRNA (1
.mu.g/.mu.l in TE), 2 .mu.l Xho(dT) primer (1.4 .mu.g/.mu.l), and 3
.mu.l water was combined, and the mixture was incubated at
70.degree. C. for ten minutes, quickly spun, and placed on ice.
Second, 1 .mu.l RNasin (40 U/.mu.l), 5 .mu.l 5.times. Reaction
Buffer (BRL), 2.5 .mu.l 0.1M DTT, and 1.5 .mu.l dNTP mix was added,
and the mixture was incubated at room temperature for ten minutes.
After incubation, 5 .mu.l of Superscript/MMLV RT mix (1:1) was
added, and the mixture (25 .mu.l total volume) was incubated at
room temperature for five minutes followed by a 90 minute
incubation at 40.degree. C.
[0100] After completion, 3.2 .mu.l of 0.5 M EDTA (pH 8.0) was added
to the radioactive reaction, and then the radioactive and
nonradioactive reactions were combined. For subtractive
hybridizations, it was necessary to remove the cRNA template by
alkaline hydrolysis. This was done by adding 25 .mu.l of TE (pH
7.5) and 5.8 .mu.l of 2 M NaOH. This resulted in a 20 mM final
concentration of EDTA and a 138 mM final concentration of NaOH. The
mixture was heated for 30 minutes at 68 to 70.degree. C., and then
12.2 .mu.l of 1 M Tris (pH 7.5) and 5.8 .mu.l of 2 N HCl was added
to neutralize the reaction. The final volume was 100 .mu.l of which
2 .mu.l was removed and counted to determine the percent
incorporation of .sup.32P-dCTP into cDNA. This analysis revealed
that 7000 ng of cRNA was converted to 2598 ng of first strand cDNA.
This first strand cDNA was subtracted against adult rat brain and
liver polyA.sup.+ RNA.
[0101] For the subtractive hybridizations, the first strand cDNA
was chromatographed on a sephadex G-50 column (NICK, Pharmacia) to
remove unincorporated dNTPs, especially the unincorporated
.sup.32P-dCTP in order to allow the efficiency of subtraction to be
followed. After the cDNA was eluted from the NICK column, it was
mixed with 60 .mu.g of adult rat brain polyA.sup.+ RNA that was
coupled to biotin (2.times. Bio RNA). The cDNA and polyA.sup.+ RNA
mixture was precipitated by adding {fraction (1/10)}th volume 3M
NaOAc (pH 5.2) and 2 volumes 100% ethanol. This mixture then was
pelleted and resuspended in 20 .mu.l TE (pH 7.5) and 20 .mu.l
2.times. Subtraction Hybridization Buffer (100 mM Hepes (pH 7.6),
0.4% SDS, 4 mM EDTA, 1 M NaCl). The resuspended cDNA and
polyA.sup.+ RNA mixture was then incubated at 95.degree. C. for two
minutes, quickly spun, and submerged in a 60.degree. C. water bath
for 48 hours to allow hybrids to form between the cDNA and
biotinylated polyA.sup.+ RNA (BioRNA).
[0102] The cDNA/BioRNA complexes were removed as follows. First, 40
.mu.l 1.times. Subtraction Hybridization Buffer lacking SDS and 20
.mu.l Strepavidin (1 mg/ml) was added, and the resulting mixture
incubated at room temperature for ten minutes. After incubation,
the cDNA/BioRNA complexes were removed by extraction with
phenol/chloroform/isoamyl alcohol. The phenol phase was
back-extracted with 1.times. Subtraction Hybridization Buffer
lacking SDS, and the aqueous phases pooled. Once pooled, 20 .mu.l
Strepavidin (1 mg/ml) was added, and the resulting mixture
incubated at room temperature for ten minutes. After incubation,
remaining cDNA/BioRNA complexes were removed by extraction with
phenol/chloroform/isoamyl alcohol. The phenol phase was
back-extracted with 1.times. Subtraction Hybridization Buffer
lacking SDS, and the aqueous phases pooled. The pooled aqueous
phases (about 400 .mu.l) were extracted with chloroform/isoamyl
alcohol. At this point, an aliquot of the aqueous phase was counted
to determine the amount of cDNA remaining. Results revealed that
78% of the starting cDNA was removed with 22% remaining (572
ng).
[0103] To perform a second round of subtraction, the aqueous phase
(about 400 .mu.l) containing the non-hybridizing first strand cDNA
was mixed with 30 .mu.g of adult rat brain polyA.sup.+ RNA coupled
to biotin and 30 .mu.g of adult rat liver polyA.sup.+ RNA coupled
to biotin. The cDNA and biotinylated polyA.sup.+ RNA was
co-precipitated and hybridized as described for the first round. In
addition, the cDNA/BioRNA complexes were removed as described
above, and the percentage of non-hybridizing cDNA remaining was
determined. Results revealed that two rounds of subtraction removed
87.5% of the starting cDNA with 12.5% of the starting cDNA
remaining.
[0104] A third round of subtraction similar to the second round was
performed using the remaining cDNA. Analysis of the remaining cDNA
revealed that the three rounds of subtraction had removed 90% of
the starting cDNA leaving 10% of the starting cDNA (255 ng).
[0105] The remaining single stranded cDNA was used to synthesize
double stranded cDNA for the subtracted cDNA library. First, the
single stranded cDNA (300 .mu.l) was alkali treated to remove any
remaining RNA as follows. The final concentration of EDTA was
adjusted to 20 mM by addition of 13 .mu.l of 0.5M EDTA, and then 20
.mu.l of 2M NaOH (120 mM final concentration) was added. This
mixture was incubated at 68.degree. C. for 30 minutes and then
neutralized by adding 40 .mu.l 1 M Tris (pH 7.5) and 20 .mu.l 2 N
HCl. The cDNA was precipitated by adding 10 .mu.l glycogen (10
mg/ml), {fraction (1/10)}th volume 3M NaOAc (pH 5.2), and 2 volumes
ethanol. The cDNA then was pelleted, resuspended in 100 .mu.l of TE
(pH 7.5), and purified on a sephadex G-50 column (NICK, Pharmacia).
The purified cDNA was re-precipitated using glycogen, pelleted, and
resuspended in TE (pH 7.5) as described. Second, 50 .mu.l
resuspended cDNA (single stranded, subtracted cDNA), 20 .mu.l
5.times. Sequenase Buffer, and 13 .mu.l water was combined, and the
mixture incubated at 65.degree. C. for five minutes, 37.degree. C.
for ten minutes, and room temperature for 30 minutes. After
incubation, 5 .mu.l dNTP mix, 5 .mu.l 0.1 M DTT, 2 .mu.l Sequenase
(13 U/.mu.l), and 2 .mu.l Klenow (5 U/.mu.l) was added, and the
mixture (100 .mu.l final volume) incubated at 37.degree. C. for one
hour. The dNTP mix contained 10 mM dATP, 10 mM dCTP, 10 mM dGTP,
and 10 mM dTTP. The reaction was terminated by adding 3 .mu.l of
0.5 M EDTA (pH 8.0) followed by two extractions with
phenol/chloroform/isoamyl alcohol and a final extraction with
chloroform/isoamyl alcohol. The double stranded cDNA was ethanol
precipitated, pelleted by centrifugation, and resuspended in 86
.mu.l TE (pH 7.5).
[0106] The double stranded cDNA was then restriction digested as
follows. Eighty-six (86) .mu.l cDNA, 10 .mu.l 10.times. EcoRI
Reaction Buffer (NEB), 2 .mu.l EcoRI (20 U/.mu.l), and 2 .mu.l XhoI
(20 U/.mu.l) was combined, and the mixture (100 .mu.l final volume)
incubated at 37.degree. C. for one hour. After this incubation, an
additional 2 .mu.l EcoRI (20 U/.mu.l) and 2 .mu.l XhoI (20 U/.mu.l)
was added, and the mixture again incubated at 37.degree. C. for one
hour. After digestion, the reaction was extracted twice with
phenol/chloroform/isoamyl alcohol-followed by one
chloroform/isoamyl alcohol extraction. The digested cDNA was
precipitated with ethanol, pelleted by centrifugation, and
resuspended in 40 .mu.l of 10 mM Tris (pH 7.5), 1 mM EDTA, 100 mM
NaCl, and 20 .mu.l loading buffer. The cDNA was divided into two
aliquots, and each aliquot was size-fractionated on a 1 ml BioGel
A-50 m column. The columns were rinsed with 10 mM Tris (pH 7.5), 1
mM EDTA, and 100 mM NaCl, with 50 .mu.l fractions being collected.
One column was run to select for only relatively long cDNAs while
the other was run to select for all cDNAs. These separate pools
were then extracted twice with phenol/chloroform/isoamyl alcohol
followed by one chloroform/isoamyl alcohol extraction. The cDNA was
precipitated by adding 5 .mu.l yeast tRNA (1 .mu.g/.mu.l) and 2
volumes of 100% ethanol. The cDNA was pelleted by centrifugation
and directionally cloned into lambda phage UniZAP as follows. For
the regular cDNAs (all sizes), 4 .mu.l water, 2 .mu.l 5.times.
Ligase Buffer (BRL), 2 .mu.l UniZAP (500 ng/.mu.l), and 2 .mu.l T4
DNA Ligase (10 U/.mu.l) was added to the pelleted cDNA, and the
mixture (10 .mu.l final volume) incubated at 14.degree. C.
overnight. For the large cDNAs, 2 .mu.l water, 1 .mu.l 5.times.
Ligase Buffer (BRL), 1 .mu.l UniZAP (500 ng/.mu.l), and 1 .mu.l T4
DNA Ligase (10 U/.mu.l) was added to the pelleted cDNA, and the
mixture (5 .mu.l final volume) incubated at 14.degree. C.
overnight. The ligated cDNA was then packaged using packing
extracts (Stratagene) and titered on XL1-Blue MRF cells. The
subtracted 3 hr MECS/CHX cDNA library containing large cDNAs
(designated IEG-Lg cDNA library) had 239,000 recombinants, and the
subtracted 3 hr MECS/CHX cDNA library containing regular cDNAs
(designated IEG-Reg cDNA library) had 4,992,000 recombinants. A
portion of each library was rescued as pBluescript plasmid, and the
cDNA inserts analyzed. Of 46 plasmids analyzed from the IEG-Lg cDNA
library, all contained cDNA inserts with the average insert size
being 1.36 kilobases. Of 44 plasmids analyzed from the IEG-Reg cDNA
library, 43 contained cDNA inserts with the average insert size
being 0.9 kilobases.
[0107] Duplicate southern blots containing cDNA from the 44
plasmids analyzed from the IEG-Reg cDNA library were probed with
control and stimulated subtracted .sup.32P-oligolabeled cDNA from
rat hippocampus. Eleven of the 44 cDNA inserts gave a clear
differential signal that was stronger with the 3 hour MECS/CHX cDNA
probe than with the control cDNA probe. This result indicates that
1 in 4 of the clones in the IEG-Reg cDNA library is derived from an
IEG.
Example 2
Preparation of Subtracted cDNA Probes
[0108] Subtracted cDNA was prepared using exactly the same protocol
described in example 1 with the exception that rather than in vitro
cRNA being used as the template for cDNA synthesis, polyA.sup.+ RNA
derived from control rat hippocampi or rat hippocampi from rats
treated with the 3 hour MECS protocol was used. After first strand
cDNA synthesis, the RNA template was denatured by alkaline
hydrolysis, and the free nucleotides removed by chromotography on
sephadex G-50 (NICK, Pharmacia). The cDNA was precipitated using
{fraction (1/10)}th volume 3M NaOAc (pH 5.2), 2 .mu.l glycogen (20
mg/ml), and 2 volumes ethanol, pelleted by centrifugation, and
resuspended in TE (pH 7.5). The final concentration was 25
ng/.mu.l. The single strand of cDNA was labeled to high specific
activity (2-4.times.10.sup.9 cpm/.mu.g) by oligolabelling
(Pharmacia) with .sup.32P dCTP (3000 Ci/mmole). Free nucleotides
were removed by chromotography on sephadex G-50 (NICK column,
Pharmacia), and the purified .sup.32P-labeled subtracted cDNA used
to probe the subtracted cDNA libraries.
Example 3
Screen Subtracted Libraries
[0109] The IEG-Reg and IEG-Lg cDNA libraries were plated on NZCYM
agarose plates at a density of 500-800 plaques/plate. Duplicate
nitrocellulose filter lifts were prepared from each plate using
standard techniques. The filters were prehybridized overnight at
68.degree. C. in 5.times.SSPE (pH 7.4), 10% dextran sulfate, 0.2%
SDS, 5.times. Denhardt's Solution, and 50 .mu.g/ml boiled,
sonicated salmon sperm DNA. The first lift from each plate was then
hybridized with 4.times.10.sup.6 cpm/ml of the control subtracted
cDNA probe and the second lift with 4.times.10.sup.6 cpm/ml of the
3 hour MECS stimulated subtracted cDNA probe. Hybridization was
done in freshly prepared 5.times.SSPE (pH 7.4), 10% dextran
sulfate, 0.2% SDS, 5.times. Denhardt's Solution, and 100 .mu.g/ml
boiled, sonicated salmon sperm DNA at 68.degree. C. for three days.
Filters were washed twice at room temperature for 30 minutes in
2.times.SSC/0.2% SDS, twice at 60.degree. C. for two hours in
0.5.times.SSC/0.2% SDS, and then dried and exposed to X-Ray film
for one to seven days. Clones exhibiting greater hybridization
signals with the stimulated cDNA probe than those observed with the
control cDNA probe were picked for further analysis.
[0110] The putative neuronal IEGs were analyzed by reverse northern
analysis and northern analysis to confirm that they were true
differentially hybridizing cDNAs. The nucleotide sequence from the
ends of these cDNAs was determined, and those sequences not
matching the sequences of known genes were used to obtain
full-length cDNAs from cDNA libraries.
Example 4
Construction of a cDNA Library Enriched for Near Full-Length IEG
cDNAs
[0111] Since the initial isolates for all of the IEGs represented
small cDNAs derived from the 3' regions of the corresponding RNA,
it was necessary to rescreen other libraries to obtain full-length
or near full-length cDNAs. For this purpose, a cDNA library
enriched for neuronal IEGs with very long inserts was prepared from
3 hour MECS/CHX polyA.sup.+ RNA isolated from rat hippocampi. This
RNA was already relatively enriched for neuronal IEGs since the
MECS/CHX stimulus produces a large induction of IEG expression.
Further, the cDNA was synthesized in the presence of methylmercuric
hydroxide to eliminate RNA secondary structure allowing for the
synthesis of long cDNAs using Superscript II Reverse Transcriptase
(BRL).
[0112] The basic protocol used to synthesize cDNA was as follows.
First, RNA secondary structure was denatured with methylmercuric
hydroxide which forms adducts with imino groups of uridine and
guanosine in the RNA and disrupts Watson-Crick base pairing.
Briefly, 22 .mu.l polyA.sup.+ RNA (0.5 .mu.g/.mu.l in either 10 mM
Tris/1 mM EDTA (pH 7.0) or water) was incubate at 65.degree. C. for
five minutes and then cooled to room temperature over five minutes.
Once cooled, 2.2 .mu.l 100 mM CH.sub.3HgOH (90 .mu.l depc'd water
plus 10 .mu.l 1 M CH.sub.3HgOH) was added, and the mixture
incubated at room temperature for one minute. After incubation, 4.4
.mu.l 700 mM 2-mercaptoethanol (190 .mu.l depc'd water plus 10
.mu.l 14 M 2-mercaptoethanol) was added, and the mixture (final
volume 28.6 .mu.l) incubated at room temperature for five
minutes.
[0113] Second, the first strand of cDNA was synthesized as follows.
The volume of the denatured RNA mixture was adjusted by adding 26.4
.mu.l water such that the concentration of RNA was 0.2 .mu.g/.mu.l.
In the radioactive reaction, 5 .mu.l (1 .mu.g) polyA.sup.+ RNA, 2
.mu.l 10.times. Strand 1 Buffer (Stratagene), 1.2 .mu.l Strand 1
dNTP mix.(Stratagene), 0.8 .mu.l Xho/dT linker primer (1.4
.mu.g/.mu.l), 5 .mu.l water, 3 .mu.l dCTP.sup.323000 Ci/mmole
(NEN), and 1 .mu.l RNase Block (Stratagene) was combined, and the
mixture (final volume 18 .mu.l) incubated at room temperature for
ten minutes to allow the primer to anneal to the RNA. In the
nonradioactive reaction, 25 .mu.l (5 .mu.g) polyA.sup.+ RNA, 5
.mu.l 10.times. Strand 1 Buffer (Stratagene), 3 .mu.l Strand 1 dNTP
mix (Stratagene), 2 .mu.l Xho/dT linker primer (1.4 .mu.g/.mu.l), 9
.mu.l water, and 1 .mu.l RNase Block (Stratagene) was combined, and
the mixture (final volume 45 .mu.l) incubated at room temperature
for ten minutes to allow the primer to anneal to the RNA. After the
room temperature incubation, 2 .mu.l and 5 .mu.l of reverse
transcriptase mix (4 .mu.l Superscript II (BRL 200 U/.mu.l) plus 1
.mu.l MMLV RT (Stratagene)) was added to the radioactive and
nonradioactive reactions, respectively. The reactions then were
incubated at 40.degree. C. for one hour and placed on ice. Two
.mu.l of cDNA was removed from the radioactive reaction and added
to 18 .mu.T.sub.10E.sub.1 and 2 .mu.l 0.5M EDTA. Two (2) .mu.l of
this mixture then was applied to a PEI strip to determine the
percent incorporation and quantity of cDNA synthesized, while 18
.mu.l was mixed with sample buffer and ran on a gel to assay cDNA
quality.
[0114] Third, the second strand of cDNA was synthesized as follows.
Both the radioactive and nonradioactive reactions were kept on ice
to prevent "snapback" cDNA synthesis. For the radioactive reaction
(18 .mu.l), 10 .mu.l 10.times. Second Strand cDNA Buffer, 3 .mu.l
Second Strand dNTP mix, 62.5 .mu.l water, 1 .mu.l RNaseH (1.5
U/.mu.l), and 5.5 .mu.l DNA Polymerase I (9 U/.mu.l) was added, and
the mixture (100 .mu.l final volume) incubated at 16.degree. C. for
2.5 hours. For the nonradioactive reaction (50 .mu.l), 20 .mu.l
10.times. Second Strand cDNA Buffer, 6 .mu.l Second Strand dNTP
mix, 111 .mu.l water, 2 .mu.l RNaseH (1.5 U/.mu.l), and 11 .mu.l
DNA Polymerase I (9 U/.mu.l) was added, and the mixture (200 .mu.l
final volume) incubated at 16.degree. C. for 2.5 hours. Four (4)
.mu.l of cDNA was removed from the radioactive reaction and added
to 18 .mu.l T.sub.10E.sub.1 and 2 .mu.l 0.5M EDTA. Two 1 .mu.l of
this mixture then was applied to a PEI strip to determine the
percent incorporation and quantity of cDNA synthesized, while 18
.mu.l was mixed with sample buffer and ran on a gel to assay cDNA
quality.
[0115] The cDNA from both the radioactive and nonradioactive
reactions were extracted twice with phenol/chloroform/isoamyl
alcohol followed by one extraction with chloroform/isoamyl alcohol.
After extraction, the cDNA was precipitated with 100% ethanol,
pelleted by centrifugation, and resuspended in 39.5 .mu.l water. To
blunt the cDNA ends, 5 .mu.l 10.times. T4 DNA Polymerase Buffer
(NEB), 2.5 .mu.l dNTP mix (2.5 mM each dNTP), and 3 .mu.l T4 DNA
Polymerase (3 U/.mu.l) was added to the 39.5 .mu.l of cDNA, and the
mixture (50 .mu.l final volume) incubated at 16.degree. C. for 30
minutes. After incubation, 350 .mu.l TE (pH 7.5) was added, and the
mixture (400 .mu.l final volume) extracted twice with
phenol/chloroform/isoamyl alcohol followed by one extraction with
chloroform/isoamyl alcohol. After extraction, the cDNA was
precipitated with 100% ethanol, pelleted by centrifugation, and
resuspended in 17 .mu.l water.
[0116] EcoRI/NotI adaptors were ligated to the cDNA, allowing for
the quick identification of artifactual cDNAs generated by the
ligation of two independent cDNAs prior to ligation into the lambda
phage vector. To ligate the EcoRI/NotI adaptors to the cDNA, 3
.mu.l 10.times. Ligase Buffer, 4 .mu.l EcoRI/NotI Adaptors (1
.mu.g/.mu.l), 3 .mu.l 10 mM ATP, and 3 .mu.l T4 DNA Ligase (400
U/.mu.l) was added to the 17 .mu.l cDNA, and the mixture (30 .mu.l
final volume) incubated at 10.degree. C. overnight. After the
overnight incubation, 1 .mu.l T4 DNA Ligase and 1 .mu.l 10 mM ATP
was added, and the mixture (32 .mu.l final volume) again incubated
at 10.degree. C. overnight. After this second overnight incubation,
270 .mu.l TE (pH 7.5) was added and the mixture extracted twice
with phenol/chloroform/isoamyl alcohol followed by one extraction
with chloroform/isoamyl alcohol. After extraction, the cDNA was
precipitated with 100% ethanol, pelleted by centrifugation, and
resuspended in 30 .mu.l water.
[0117] To kinase the cDNA ends, 4 .mu.l 10.times. T4 Polynucleotide
Kinase Buffer, 4 .mu.l 10 mM ATP, and 2 .mu.l T4 Polynucleotide
Kinase (10 U/.mu.l) was added to the 30 .mu.l of cDNA, and the
mixture (40 .mu.l final volume) incubated at 37.degree. C. for 30
minutes. After incubation, 2 .mu.l T4 Polynucleotide Kinase was
added, and the mixture (42 .mu.l final volume) incubated at
37.degree. C. for 30 minutes. After this second 30 minute
incubation, 170 .mu.l TE (pH 7.5) was added, and the mixture
extracted twice with phenol/chloroform/isoamyl alcohol followed by
one extraction with chloroform/isoamyl alcohol. After extraction,
the cDNA was precipitated with 100% ethanol, pelleted by
centrifugation, and resuspended in 85 .mu.l water.
[0118] To digest the 3' cDNA ends with XhoI, 10 .mu.l 10.times. NEB
Buffer #2 and 5 .mu.l XhoI (20 U/.mu.l) was added to the 85 .mu.l
of cDNA, and the mixture (100 .mu.l final volume) incubated at
37.degree. C. for 45 minutes. After incubation, 3 .mu.l XhoI (40
U/.mu.l) was added, and the mixture (103 .mu.l final volume) again
incubated at 37.degree. C. for 45 minutes. After this second
incubation, 120 .mu.l TE (pH 7.5) was added, and the mixture
extracted twice with phenol/chloroform/isoamyl alcohol followed by
one extraction with chloroform/isoamyl alcohol. After extraction,
the cDNA was precipitated with 100% ethanol, pelleted by
centrifugation, and resuspended in 20 .mu.l 10 mM Tris (pH 7.5), 1
mM EDTA, 100 mM NaCl, and 5 .mu.l loading buffer. This resuspended
cDNA then was size-fractionated on a 1 ml BioGel A-50 m column to
select large cDNAs. The column was rinsed with 10 mM Tris (pH 7.5),
1 mM EDTA, and 100 mM NaCl. Thirty-six (36) fractions (50
.mu.l/fraction) were collected. Aliquots from individual fractions
were electrophoreses on 1% agarose to identify fractions containing
cDNAs longer than 2 kilobases. Such fractions were pooled, and the
resulting mixture of pooled fractions was extracted twice with
phenol/chloroform/isoamyl alcohol followed by one extraction with
chloroform/isoamyl alcohol. After extraction, the cDNA was
precipitated by adding 2 .mu.l glycogen (20 mg/ml) and 2 volumes
100% ethanol, pelleted by centrifugation, and resuspended in 5
.mu.l water.
[0119] To directionally clone the cDNA into UniZAP, 2 .mu.l UniZAP
(500 ng/.mu.l), 1 .mu.l 10.times. T4 DNA Ligase Buffer, 1 .mu.l 10
mM ATP, and 1 .mu.l T4 DNA Ligase (4000 U/.mu.l) was added to the 5
.mu.l of cDNA, and the mixture (10 .mu.l final volume) incubated at
12.degree. C. overnight. After incubation, the cDNA was packaged
into phage particles. To package the cDNA, the ligation reaction
(10 .mu.l final volume) was divided into two packaging reactions
with each containing 5 .mu.l of ligation reaction together with a
packaging extract (Stratagene). This mixture was incubated at
22.degree. C. for 2 hours. After incubation, the two reaction
mixtures were pooled and the library titered on IL1-Blue MRF
cells.
[0120] This 3 hr MECS/CHX library (designated IEG-FL 3 hr MECS/CHX
cDNA library) had a titer of 4.4.times.10.sup.6 primary phage. The
library was amplified and used to isolate full length cDNAs derived
from novel neuronal IEGs. The relative abundance of near full
length neuronal IEG cDNAs in this library was substantially higher
than the levels experienced using other cDNA libraries. In a single
cDNA library screen, full length cDNAs for four different novel
IEGs were obtained. Three of the four IEG cDNAs were derived from
mRNAs of 4 kilobases, while one was derived from an mRNA of 3
kilobases.
[0121] The nucleic acid sequencing of the IEG cDNAs was performed
at Johns Hopkins School of Medicine and at Applied Biosciences,
Inc., CA using the Sanger method with fluorescent dye
termination.
[0122] Northern blot analysis was performed both to confirm that
the cloned cDNAs represent tissue mRNA that is rapidly induced by
brain activation and to assess the size of the mRNA transcript. The
latter is essential information for the identification of authentic
full length clones. Either 20-25 .mu.g of total RNA or 2 .mu.g of
polyA.sup.+ RNA was sized by denaturing agarose gel chromatography
and transferred to nitrocellulose. Blots were then hybridized with
[.sup.32P] labeled cDNAs. Labelling was done using the random
primer method (Pharmacia).
[0123] In addition, in situ hybridization was performed both to
confirm that the cloned cDNAs represent tissue mRNA that is rapidly
induced by brain activation and to confirm that the mRNA was
induced in activated neurons. In situ hybridization was performed
as described previously (Andreasson and Worley, Neuroscience 69:
781-796 (1995)).
Example 5
IEG Nucleic Acid
[0124] The following clones were identified as being IEG nucleic
acid as described in Example 3. In addition, certain clones were
identified by chip-hybridization between PCR fragments generated
from rat hippocampus ESTs and .sup.32P-dCTP-labeled cDNA derived
from polyA.sup.+ RNA of rat hippocampus from MECS treated animals
and controls.
[0125] One IEG nucleic acid clone was designated A003. The first
library screen produced a fragment (A003-1-1) of 1.6 kilobases (kb)
with a polyA sequence at the 3'-end. A second round of screening
was performed using a probe prepared from the 5'-end of A003-1-1.
This screen produced two clones: A003-1 (2.8 kb) and A003-2 (1.3
kb). The fragments from the secondary screen were sequenced from
both ends. These fragments formed a contig at their 3'-end with the
A00-3-1-1 fragment. The following two nucleic acid sequences are
within the A003 clone:
1 5'- (SEQ ID NO:1) TTGCAGATCAGCACCTTTTGATGATGCCTG-
CCCAACAGTGGGTAATGCTNACAGCAA AGCACCACTTTACGCTTTTTAGTTGTGCTG-
GGTTCATGGCTGGACATACACCAACCA GCCTTGACCCCACAGGAATGCCAAGTTGGC-
TGGAATGTAACCCAACCTAGTTTCTGC GCTTCGCTCCTCTCCCAGTGCAAGGTGCTA-
AACACCCACTCACAAGCCTGCTGTCAA GCTGCGACCTTGGGGGCTGGTTAGAAAGGG-
CTGCCTCCTTCCAGCAATAGAAGTTCA TGAATTTGAGGCTGGAGATAGGTCAAGACC-
ACTGTGATAACTATAAAGACTGTAGC AGCCACAAAGGAGACCCCCAAATAACTGGAG-
GCATGGGCACTGACGTACCAGATGA GGTTATGTTTGGAGCTGAAGGCTTGCTCTGTG-
CTTCTTGGTAGCATCTTTTGTCCTCT TGGGACATGGTTGACCCCATACTGTCCACTG-
AGCTTGGGAGATGACAGTTGAATAAA AAAAAAAAAAAAAAA-3' and
5'-CGGCTTAATTAACCCTCACTAAA (SEQ ID NO:2)
GGGAACAAAAGCTGGAGCTCCACCGCGGTGGCGGCCGCTCTAGAACTAGTGGATCC
CCCGGGCTGCAGGATTCTGCGGCCGATTAAGAAGCCTGCTGATGTCCTTAGGCGAGG
ACATTAACTCCAGTCTCTGACAGACTTTGGACATCCAGAATAAGTTCTTTTTGTATAT
CAGAGCACAGAGCCCAGCTTTAGCCTCTGATGGACCTCAGGAACCAAGAAGGAGGG
ACTTCCTTAACATTCTAGAGATGGGACTCTAACTCTAGCTCTTGTGTTAAGCCCTGAA
GTCCAGAAAGAAGTAGTTCTTTGACATTCTAGTGCCAAGATCCAGCCTCTAAGAGAA
CTCTGATGTCTAAAGAAAGTCTTTCATAGTCTAGNCCAGTCACCAGTGAAGCTAAAC
ACCTGAAAACTATTAGATTCTCTGGAGCCAGGAATCCATCTCAAGTCTCTCATAAAG
CCCAAATGTCCCAGGAGAAGTTGACAATATAAAGCCGTATCTCGATGGACTTTTGAA
GAAGCTCAGAAAAGGAGACCACCTTGGTAGTCTTGATCTAGGACTCTGGCTTGTTTG
TCTCCAGGGACGTTTACATGTATAAAAAGAGGGACCTTTCTGATGATTCAGAACTGG
GACTCCACCTCCATCCTTTGATGAAAGCTCAAATGTCCAGAAAGAGGGGCCTCTCTG
ATATTCTAGAGTAGGACCCTCCCTCCAGCCTTTGATGGTGTCCAGATGTCCAGAAAG
AGGGGCCTCTCTGATGTTCCAGACCTAGGGCCCTCCCTCCAGCCTTTGATGGTGTCC
AGATGTCCAGAAAGAGGGACTTCTCTGATGTTCCAGACCTAAGACTCTAGCTCCAGC
CTTTGATGAAGCTCAGATGTTCAGAAAGGGGGGCCTCCATGATGTTCTAGAACCAGG
ACTCCACCTCTAGCCTTTGATGGTGTCCAGATGTCCAGAAAGAGGGTCTTCCATGAT
TTCTAGGACCAAGACTTTACCTCCAGCCTTCTATGCCTCCATGTCTCCAGTAAAGCTT
AGGTGTCCAGAAAAGAGCATTCTCAATGAATTTATAGAACCAGGACTCTTTCTCCAG
CCTTTGATGACGTTCAGATGTTCATAAAGAAGAACTTCCACAATGTACTAAAGCTAT
GACTCCATCTCCATCCTTTGATGAAAAGGGACTTCCTTCCACTCTGTTCCAGAAGCCT
AGCTCCACCTCTAATCTTTGTTGATGTCCAATTATCCAGAAAGAGGGGGCCTTTAGA
ACAAAGACTGTACTTTTATTCATTGATAAAGCACAGATTCCAGAAGCACAGAAATCT
AGAAAGAGGGTCCTCCCTAACACGCTCGAGCTAGAACCCCGGTGCAAGGGTCTGAA
ACTTAGACACCAGAAGACCGCTTTGTCCTACAACAAGTCTGCATTTTCTAAATCTCC
AGGTGGCTGAT: CAGAAGGGTCCAGGAAGGTATGGGG-3'.
[0126] Northern blot analysis using the 3'-end of A003 revealed the
presence of two mRNA transcripts. The more abundant transcript was
2.2 kilobases in length, while the less abundant transcript was 4.8
kilobases in length. This analysis also revealed that the
expression of A003 mRNA was marginally upregulated in response to
the multiple MECS treatment. The multiple MECS treatment involved
the induction of multiple maximal electroconvulsive seizures
followed by the preparation of total RNA from rat hippocampus four
hours post-seizure. This multiple MECS treatment was designed to
mimic ischemia.
[0127] Another IEG nucleic acid clone was designated A013. The
first library screen produced the clone designated A013-8. The
5'-end of A013-8 was used as a probe for the second round of
screening. This second screening produced two additional clones:
A013-4 and A013-26. The A013-8, A013-4, and A013-26 clones were
sequenced using either the gene specific primer used to generate
the probe for the second round of library screening, or the T3 and
T7 primers. Both A013-4 and A013-26 made a contig on their 3'-ends
with the A-013-8 clone. In addition, the sequence from the 5'-ends
of A013-4 and A013-26 revealed that they from contigs between each
other. Further, the sequence data from the 5'-ends of A013-4 and
A013-26 revealed the presence of an open reading frame of at least
720 basepairs (bp) Based on the combined length of the obtained
clones, the A013 clone is at least 3.0 kb in length. The following
two nucleic acid sequences are within the A013 clone:
2 5'-GGCACGAGATCACTCAGTGTCTTCACTGAAC (SEQ ID NO:3)
CAAATCGTCNTTTTTACAGAGAGATGCAAAGCTTCAGCGAAGACATTTAGCTTTTT
AAAATGTATAATTCCTGTGGCTACATATGCAAGTAGGGTCCCATTATGTTTTTTTTCA
TTAGTGGAAACTAATCCTTTTGTGCTGTGTTTAATCAGTATTAGCTTTATAGAATTAT
AAATGTATATTCTACTTCTTGATCAAAGAACGTAGTCGGGTATTGGTTTTAGAAGTTC
AAAGTGACACTGTATAGGGCTTTCACGGTTAATGGGATTGTTAGCAAATCTTAAGGA
CATACAGCCAATGATTATCTGAGGTTACTGGCTAACTGTTTTTCACTGAGTTACTCTG
CCTTTTTGACATTTTTATTCTTTGTTTGTCAGAATCCAGAGCTTCAGGAGCCCAAATT
TTTTTATWCCGTATATATATATATATATAAATATCCATAAGCCTGGTGGATTTGTATG
CAATGCACTGCATCTATGTATTCTGATAGCATCTCATTGATTTTTGTTTGAAATAGAA
AGAAAGATAGTATCCCAAATGAGTTATCTTTAACAGAAAGCTGAGTTTAACTTTTAT
TACCTATATAATAATTGATATTGCCAATTACCATTCTGAATTTCATATAGTATAAGTT
AGACATTGCTTAATCCCCTTTTAAATGTATTTACATAGACATGAACACTCAAATTGCT
GGATTTTTTAAATATATCTGACATAATTTTTTTCATCTGTTACATTCAAGTTAGCTTGT
TTAGCCCAGATTTCAGAATAGTAAAGGAGGAAAGGAACCGCATTCCAGGGAAACCT
CTGAGGCCAAGTCAGAGTCCAGAACTGTAAACACACAGGCCTGCAAGCCAACATTA
GTCGTGAAATCCCTAACACGTCACTGGATTCTCTCTGTCAGCGCAAGTGTCAGCTGC
CAAAGAATAGACTTACATGAAGAAGTGCCCACATGCTGGCAGGGGCTGGCCGGCTC
CGGCCAGCAGACACTGCTAGATTGTAATATTTAAGGTCGAGTTTCGACCTGTGGTAC
ACAGCTGTGCTGTGCTCAGTCAGCAACCTCAGAACTCTGAAAAAAACATAAAAAAG
AAAAAAAAAAAAAAAAAAMATGCASCTGKYTCACTTGTGAATAGTGAATGTAAAG
GAAAGAAAGGAAAACCAAAAGCTTGTTCCATCACAGGTATGAGCTGCTATGATTCA
TGAAGAACATTCCATGGAGTATGTTTTAAAACCTTGTTATATCTGAGAGGCTTTAAA
AGCCAACTTAACTGTTTCAGGGCAACCGCGGTACAGACGTGGTCTCTGTGAGACTTC
CACCTGACCCAAGTTTTAAGTGGTACGAATGTTGTGCATTTAATGTTAAGGACAGTC
TGCAATAATAAGTAAGTAGCCAGCGTGGGTGCCCAGCAGTGCTGAGACCTGGCTGC
TCTATTGTACGCTTTGGAAACACAATTTATGCAACAGATGTCCAGATATGATTCTATT
TATGGAAAAAGTTTATATGTTTTACAAATGGTTTTACCATCTTATATTAAATGACCTT
TTGACAGGTGTGCACTGTTTTGTCTCCAGTGAGCACATACCATGCGGATTTTATATGT
ACATCAGTAGTGTGAATCCACTGGCACAGTGTGTGTAAATGCCAGATGTGGTGAGAT
TTTATCTTGTATATGTGATCAGATAAAATAACTCCTGACAGAAACTGTAAGGRAACC
CAGCTGAATGGTTTGACCTGGATGRCYKRKRTKGTWTGGTTTATGTTAAATGTATAT
TCTTTTAATCAATGAATAAAGCATTAAAAAATGGGAAAAAAAAAACTCGTGC-3' and
5'-TCTGCGGCCGCAGCATCCGGAACAACAGGAACCTCCAGAA (SEQ ID NO:4)
GTTTAGTCTTTTTGGAGATATAAGTGTCGTTCAGCAGCAAGGA- AGTCTGTCCAGCAC
ATACCTCAGCAGAGTAGACCCTGACGGCAAGAAGATTAAGCAA- ATTCAGCAGCTGT
TTGAAGAGATACTGAGCAATAGTAGGCAACTAAAATGGCTGTCC- TGTGGGTTTATGC
TGGAAATAGTAACCCCATCATCACTGTCGTCTCTGTCTAACTCC- ATTGCCAACACCAT
GGAACACCTGAGTTTACTGGACAACAACATTCCTGGTAACAGC- ACGCTCATCACCGC
AGTCGAACTAGAGCGCTTTGTAAATCTGCGCTCACTTGCCCTG- GATTTCTGTGACTTT
ACAGCTGAGATGGCGAGAGTCCTGACCGACAGCAACCATGTG- CCTTTGCAGCGACT
GTCTCTTCTGGTCCACAATGCTTCAGTGATGCTCAAGTCATTA- GACAACATGCCAAA
CGATGAGCACTGGAAGGCCCTGTCACGAAAGAGCTCCAGCCTC- CGGGTCTATCTAAT
GGCTTTTGATGTTAAAAGTGAAGACATGCTAAAGATTCTGTAA- ACCCAGTATACCACT
TGAGAAGGGTTCACTTTGGACAGCTACGTCACTTGTGTCTCA- AGGGGCTATTGGTTG
ATCTTATATTCCAGGCAGTATTGACCAAGGTTTCCTYAACCC- MWTTTWTATTGATGA
ATGATATGATTGATACGTCTGGTTTTCCGGATCTTAGTGACA- ACCGAAATGAAGATC
CATTGGTTTTATTGGCATGGCGGTGCACAAAGCTCACTCTTT- TGGCAATTCATGGTTA
CACCGTGTGGGCACACAACCTCATTGCCATTGCTCGTCTTC- GTGGCTYTTGACCTAA
AAGTGCTTTGGAAGTCACCSRAAGAAAGCATTGATTTTGAC- CAAGGTGAACTAGCCC
GACCAGGAATGTGGRWYCCCGTACATAACCTTTCTTGGAGC- AGGTATTCCCTGGGGC
CTTGGTCAAGTCTTGGCACG-3'.
[0128] Northern blot analysis using a sequence from the A013 clone
revealed the presence of a 3.2 kb mRNA transcript. In addition,
this analysis revealed that the expression of the A013 mRNA was
strongly upregulated in response to the multiple MECS treatment.
Specifically, A013 mRNA expression was induced 8.9 fold by the
multiple MECS treatment as determined from Northern blot data using
total RNA from rat hippocampus (Table I).
3TABLE I Fold induction of mRNA expression after multiple MECS
treatment Probe (rat cDNA) Fold induction (normalized for the S26)
A013 8.9 L094 7.3 L100 17.2 L119 17.8 R113 7.0 R286 2.4
[0129] Another IEG nucleic acid clone was designated A020. The
following nucleic acid sequence is within the A020 clone:
4 5'-TCAAACCNTATCTCGGTCATTCNTTTGAT (SEQ ID NO:5)
TNATAAGGGATTTKSCCGATKTCCGGCNTATTGGTTAAAAAWTGAGCTGATTTAACA
AAAATTTAACGCGAATTTTAACAAAATATTAACGCTTACAATTTGCCATTCGCCATT
CAGGCTGCGCAAYTGTTGGGAAGGGCNATCGGTGCGGGCCTCTTCGCTATTACGCCA
GCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTT
CCCAGTCACGACGTTGTAAAACGACGGCCAGTGAATTGTAATACGACTCACTATAGG
GCGAATTGGGTACCGGGCCCCCCCTCGAGGTCGACGGTATCGATAAGCTTGATATCG
AATTCGGCACGAGCGAAGCCAGGGCCTTGCACTTCCTAGGCAAGCGCTCTACCACTG
AGCTAAATCCCCAACCCCTTGTTTTATTTTTAAAGCAAACGAGATACATAATTTCARC
CATGATAATTTAAGATTATCTTGAACTCTAAGGAAATGTATATACTAAGCTATTAT
AGTTTTTATTTTCCCTAATTCAGTGGCATAATACCTTACCTTGAGTCGTTTACTACTT
CTTTGGTTTCTAAAAACTCTACTGCTAAATTACAATGTAAAAACATAGGGCTCGTAT
ATACTGTAGAGTGCTGTAGATGTCCTCGTCATCAACTATGCAATAACAGTCTGATCG
ACACATTTCAGGAKCGATCACTCTTTGGTGTGCTTCTTTAAATACTTTCAGAAGCTTA
GGATGTGCAAAGCAGGAAGACTGTGGGTGTAAATGTTTACTTATTTCTTTGAGAGTG
TTAGTAAGTCTTTTCDAAATTGCTTTTCTCTTCAAAATTATCGTTAACTTAAATGATA
ATTATCTTTGAGGTTAAACAGAAGCTCATTGACAAACTAAAGTGACTTTTTAGGGCA
TTCTTTGAGATCATAGTCTTATATCTTGGGGACTAAAATGTCATTAGACCCTAATAGA
CTAACTTGTATGTTTGTGTGGGGAAACGTTTTCCTCTCTCATTCAAGGTAACTGTTTG
CTGCCTGTTGTTACTTGTGTAGCATTCTAGAAAATGGCTAGGTTTTTTATAAGATTTA
AGACAATAGAAGTAGTTTTATATTATTATAGTTCTGTTGGAATGTGATCCTGAAATT
ATTACTGAAAATTAGAATTTTTATTTCGCTAATGACAACCTTGACTCTCAGAGATGC
AGTGTAAATTGATACCTCATCTTTCCGAGAGTTCAGAGCACAGGGCGGCAGTATGTG
AAGCTGCTTTTGCACTGACGCATTTTGATAAGTTTGGCTACTGTAATGGTAAAAGGC
TCCTCAGGCACTGACTGCATTTGGGTTCTTCCGATGGGGGATGATCCGTTCTCGTGGT
GCTGCTGGACTTATGCATTTTGGAGGTACTGCATGTATCTTCCACACTGCTTGACATT
TTCTCTGATCTGTGTGTTTGCACCAACTCATTAAAAGAAATATGCAGAAATATCTTCT
AATTCGTTGATCTTCGCTGTATGACAGTTATAATATTAAACACTTGGGTTGATCCACT
CTGTTTACATTTATCTTTCTAAGCGTCAGAAAGGGACTAACTTGAAATTATATCTAGA
GGCTTTGTATCATTTCAAAAATTAAATTTCCTTGGATACTTTAGGCAATATCTTAAAC
AACTTTTTAATAAATTTAAATATTTATATTTACGTAAGCTAAAATATACATGAATGTG
CTTTTTAATAAATTAAATACAGTTTATACTTATTTGCCAATTCACAAATAAAAAAAA
AAAAAAAAAAAAAAAA-3'.
[0130] This clone is similar to GLGF-domain protein Homer
(accession # U92079).
[0131] Another IEG nucleic acid clone was designated A021. The
following nucleic acid sequence is within the A021 clone:
5 5'-TTTTTTTTTTTTTTTTTTTTAARGGGRCCACCCC (SEQ ID NO:6)
ACCGSGCTAAAGGCCCAGGGGCCCCCCCCTTGGAGMCCCAGGGGTTTTGGCCCMCC
CCCTCACCCAAATGGTCTGCCAATGACCCAGGTACTCACAACATGTTCCAGGAGGAG
MCTGGGGCCAGGATTTTGACCAGAGGGTATGGGAAGGGAAAGGGGAGAAGAAATC
GACATTTATTTTTATTATTTATTTAAATGTTTACAWTTTCTTTGTGTTGTTCCAAGCC
CTGAATAGAAACAGATAGCATTAAAGGACTCTGTTCCCACCCCTTCTCTGTCTCTCTC
TCCCCCACTTGTGCTAACTTAGGATAACACTCTCTATTTCGTTTTGTTTCTAAAGTGA
TTTGTGGACTTGTGCCGTGTGAACTGCATTAAAAAGGTTCTGTTTTCAAAGATCGATT
GTCGTTCCTGTGGGGACAGTGGCTCCTAAGAAATCTGCATTGTAGGAGAAGACAATG
AAAGACCCTGGCCCTGTCTCTCAAAACTTAACTCTCTGTATGATTTAAAAAAAAATT
CCATTTACTTTACTTTGTGGTTACTTGATTTTGAGGAAGAAAATATTCAACTTTGTAT
AAAGACTAGGTATCAGGGTTTCTTTTGCAGTGGGAGTTGTATATATATCGTATTTTGG
TATATCGTAGAAACTCAAGCTTTATGCATCCGTATTTGGGATATGTCAATGACGTGC
AGTGAAATTTGCTATTAGACCCTGGAGGCAAACGAGTTGTACAAGGTTTTATGGCTC
CATGGGGANTTCTAATTTCCTTTCTGGGGACCTTTTGTCCCGTTTTTACAGTAATGGT
GAAATGGTCCTAGGAGGGTCTCTCTAGTCGAATTCTCCAGGCAGGACCACGTGCTCA
AAAAATCTTTGTATAGTTTTAAATTTTTGAGGAGTATCTCTGCTCAGAAGCATCTGTG
GTGGTGTGTGTTGCGTTGTTCTGTGTACTGTGTGTGACACAAGCCTACAGTATTTGCA
CTAAGGAAAGCTGTTTAGAGCTTGCTGCTATGGAGGGAAGAACATATTAAAACTTAT
TTTCCCTCGGGGWTTRTWCWMGTTTTATGTWCTTGTTGTCTTGTTGGCTTTCCTACT
TTCCACTGAGTAGCATTTTGTAGAATAAAATGAATTAAGATCAGMWRWRWRMAAA
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA- 3'.
[0132] This clone is similar to fra2.
[0133] Another IEG nucleic acid clone was designated A024. The
following two nucleic acid sequences are within the A024 clone:
6 5'-TCAGGCCTNAGCAATCCTCNTTAANTTTGA (SEQ ID NO:7)
NCCAAGNTTAACTCTTGGGGCGAATTCCTGTGNTTGCTTTCTTTCCCCATANTTCCAG
GCCCACAAANGGTTTCTGTGANTCCGAGAATCGGCCCACCATGCAGACCCACNGAG
AGGATTCAGAATGTGTGTGAGAGTGAGTGTGTGAGTGCGCGTGCGTGTGCTTTGTAT
GTGTGTTTATAGATGTAGGACATTAAGTTCCTTCTGACACAGGGAAGATGTGAGAAG
GATGGCCTGACATCAGATGACAAGAGGTCTTATAGCACATCTCTGGGCTTTTCCCTA
CCCAGAGAAGAGCCCCCTTTGATACAAATCAGTTGGATTTTCATATGCTTCAAAGGC
TTGATCTGTGAGTCACTCCAGTTTGGGACATAGGTCTGTCTGTGGCTTTGAGAAAAG
GTACTTTCAAAAGAGGGCTTTCCAGAGCACAGCTCACAGCCAGCTGTTAGGACCCCA
CCCTTCTCCTTTATTGTGGAGGTGACTCACAGCAGACTGACAGTGGTCAGACTGAGC
TTTCTGCTAAGGTGGTGAGGTAGCCAACACTGGCATGTCTCGGTAGTGGTTTGGGCA
AATTTCCGCAGGTCTCTTCCCCCAACCCTGCCTCTGATGAATAAAGACAATGAGTAC
AGTTCCTTAATTCAGGCTTTTGTGACTAGCTTACTACGGAACCGAAAATGGTCCCCTT
TGTACAAGCCGAGCTGTTATGGAATCACGGTGAACCAGACCCAGGTCTGTGGCACCT
GTTTGTTTTTTTTTTTTTTTTTTTTTTTTTAGCTCTCATTTCTACGGCATGCTTTCCAAG
GAACCAAAGGAGGGTCTCAGAGATGCCCCAAACATCCCAAAGTACACAAAGCTAAG
TAATCGATTGCTTACTTATTGCACAGCTAGACACGGATTTTAAGTCTATCTTAAAGCT
TTGAAGCAAGCTTAGCTTCTCAAAGGCCTAGCAGAGCCTTGGCACCCCAGGATCCTT
TCTGTAGGCTAATTCCTCTTATCCAGCGGCATATGGAGTATCCTTATTGCTAAAGAG
GATTCTGGCTCCTTTAAGGAAGTTTGATTTCTGATTCAGAGTCCTTGTTTCCCTGACT
TGCTCTGCCAGCCCTGCACCAGCTTTTTCGAAGTGCACTATGCTTGTGTTTAACTTCT
CCCAGTTTTATTTGGGCATAAAAGTTGTTGCCTTTATTTGTAAAGCTGTTATAAATAT
ATATTATATAAATATATGACAAAGGAAAATGTTTCAGATGTCTATTTGTATAATTAC
TTGATCTACACAGTGAGGAAAAAAATGAATGTATTTCTGTTTTTGAAGAGAATAATT
TTTTTCTCTAGGGAGAGGAGAGGTTACAGTGTTTATATTTTGAAACCTTCCTGAAGGT
GTGAAATTGTAAATATTTTTATCTAAGTAAATGTTAAGTAGTTGTTTTAAAAAGACTT
AATAAAATAAGCTTTTTCCTGTGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA-3' and
5'-GTGGCCCCTGCTCGCCGCATCATGGAGCGGATCCCCAGCG (SEQ ID NO:8)
CGCAACCACCTCCTACCTGCCTGCCCAAAACGCCAGGGCTGGA- GCACGGAGACCTG
TCAGGGATGGATTTTGCCCACATGTACCAAGTGTACAAGTCCAG- GCGGGGAATAAA
ACGGAGCGAGGACAGCAAGGAAACTTACAAATTGCCGCACCGGTT- GATTGAGAAAA
AGAGACGTGACCGGATTAACGAGTGCATTGCCCAGCTGAAGGATCT- CCTACCCGAA
CATCTCAAACTTACTACTTTGGGTCACTTGGAGAAAGCAGTGGTTCT- CGAGCTGACG
CTGAAGCACGTGAAAGCATTGACAAACCTAATTGATCAGCAGCAGCA- GAAAATCAT
GGCCCTGCAGAGCGGTTTACAAGCTGGTGATCTGTCGGGAAGAAATAT- TGAGGCAG
GACAAGAAATGTTCTGCTCCGGTTTCCAGACCTGTGCCCGGGAGGTACT- TCAGTACC
TGGCCAAGCATGAAAACACTAGGGACCTGAAGTCTTCCCAGCTTGTCAC- TCATCTCC
ACCGTGTGGTCTCTGAACTCCTGCAGGGTAGTGCTTTCCAGGAAACCAT- TGGACTCAG
CTCCCAAACCCGTGGACTTCAAAGAGAAGCCCAGCTTCCTAGCCAAGG- GATCAGAA
GGCCCTGGGAAAAACTGTGTGCCAGTCATCCAGAGGACTTTTGCTCCCT- CGGGCGGG
GAGCAGAGTGGTAGTGACACGGACACAGACAGTGGCTACGGAGGCGAAT- TGGAGA
AGGGTGACTTGCGCAGTGAGCAACCCTACTTCAAGAGCGATCACGGACGCA- GGTTC
ACCGTGGGAGAACGCGTCAGCACAATTAAGCAAGAATCTGAAGAGCCCCCCA- CCAA
AAAGAGCCGAATGCAGCTCTCAGATGAGGAAGGCCACTTCGTGGGCAGTGACC- TGA
TGGGTTCCCCATTTCTTGGGCCTCACCCACATCAGCCTCCCTTTTGCCTGCCCT- TCTAT
CTCATCCCACCATCGGCCACTGCCTATCTGCCTATGCTGGAGAAATGCTGGT- ATCCG
ACCTCTGTGCCACTGTTATACCCAAGCCTCAACACCTCAGCAGCAGCCCTCT- CCAGC
TTCATGAACCAGACAAGATCCAACTCCCTTGCTCTGCCCAGAAATCCCTTCT- CCCTTG
GCACATTCGTCCCTTGACTCTCAAGCCTGCTCAAGCCCTGAAGCAGATCCC- TCCTTA
AACTTAGAAACAAAGATAAACCTTGAGGGCAATCNCTGCGCCTTGCTTTCC- TTCCCA
CAATTCAAGACACAAAAGGTCTGTACTCAAAACAGAGAGATCAGCCCACCC- TGCAG
ACCCACAGAGAAGATTCAGAGTGTGTGTGAGAGTGAGTGAGTGTGCGTGCGT- GCGT
GCTTGTATGTATGTTTGTATATGTAGGACAATAAGTTCCTTCTGACACAAGGG- AGAC
ACGAGAAGGATAGCCTGACATCAGATGACAGACTGGAGGACTGTAGCACATCT- CTG
GGCGTTTCCCTACCCAGAGAAGAGCC-3'.
[0134] This clone is similar to a basic helix-loop-helix
polypeptide.
[0135] Another IEG nucleic acid clone was designated L003. The
following nucleic acid sequence is within the L003 clone:
7 5'-GCACGAGGGAGTTTATTTCCACGTCT (SEQ ID NO:9)
CTTAGGAAAGCCTCGCTTGGTTACACATGGCAATGATTGCAAGCAGATACACGTCTT
AACACCAGAGTACAGTACACACACATTGAGCTGCCCTCGTCTAACAAGCAGTTGCA
GTTTGTTTAAATGTGAATATCTATGAAACGAGCAAAGCAACTTTCCAGAGTATAGCT
TATCACAGAATAGTAACACATGGGCCGCTACTGTATCATACAGAGTACAACTCTATA
GCTTTTCATCCCCGTGTGAGCATTTCCAAATCACTCAATGAGCACCAAGCACGGACA
AGTGACTAAAAAGGCTAGTCCCAATCTCCCCGCAACCCTCGGCGGTAAGGGTAAAG
AATTTTGTTTCAAGTAAGTTTTCTCCTCGTCTCTCTCTTCTGAAGACCTGAGCAAAAC
CAACATTCTAAACCACCCCAAGATATGATACTAGAATTTAAAGGCCCGATGGCTTCA
ACCCAGAACCTTAACCTACTAGATAAAATCTCTCCGAATCTGACTCACTGATGCTGT
TAAGTCCGACAGTACAATCACATAGTACCTCTTTGATACTGTCAAAGTTGGTTTTAA
AAATGCCCTAAGAAAACCAAATCATTTTTGGGAGATGTTCTAAGCAAGCTTTCCAAC
ATATAAAGAACAAAACCATGTTACTAAAAACATGGTGCAGGTCCTCACAAAACATT
TACTGCTACTACCAGGAAACCAAGCTACTCTTGGTTTGTGCTCCTGGTGATAACTGG
TGAGCTTTGGACAGCTGCTGGCACATGTCCACTGTGTTCCGTTTTATAATCAAGTGTC
AGTTTTCCACTCGACAGAGATTAAAGACAATAGCTTAAAAGTGAAAATGAAATTTCA
AGTAGAAGCTACAATTGAATGCTACTTGTTGAGACTTTTAACTTCACATCCAAATA
TCAAAAACTTAACTTTGACGACACATGCACACAAACACACCATTTGGGAAAGGGTCT
TGTTATGCAGTTCAAGCTGGCCTTGAACTCATGATCTCCTGCCTCAGTTTCAATGGGCA
GTAGCACTGGACCTTACTGTGGGCAGAAAGTATTGCTCCAATTAGAAAGCATTACTA
TACACTTCACTTCGTCATGTGCCTAGTGTGGCTCTGAAGGCATAGGAACAATGAAAT
TAAATTCTTCAGCAGCTGAGGATTCTCTATACTTCAACATTCTGAACTTCAATCATGG
CTTCACATTTGAGGCTGAGCTAGATACAAAAATATCAAAACATCCCATAGAATTGTT
TATTTCCCTATGTTACTGTTTACCCAAGGAATGTGAAGACTAAAAAGGACTCATTTG
GTTGTTTAATTATGATTAAATTATGTAAATATACAAACATTTAACAAAGCCATCATA
TTCCAATCTTTTACGAATTCTAACTGCTAGCAGTTGAGCAGCTTTTAGATATCACTAA
TAAAATATACAATTTAAAATAGTCGCATTCAATCCTACTAACTTTATAAATAACTTCT
TAGGTTAGACTTCTTCCTGCCTAAGTTTATAAGACAGTCTAAACCCAAAACTCAACA
CATATTAAGCTTTTTAAAAACTCCATATAGTTCTAAAGTAACCTCAATGTATTCCCAA
GAACCGCCACCATCAATCAGCTCACTCCCTCACACCACTGACTTTAAGACGCTCCTG
GGTGGAGAACTGCCAGGCAGAAGCTCTACCTTTCTAGTGTGTGTGGTGGTCTGCTGC
TCCTAGTCCAGATCTGGACCACATCAGCACAGCATCAGTGTGACTCAGCACTGAGGC
CTTGAGCGCTCTTCCCCCCGATGGCCTGTGTATAGAGGTGTCTAATTCCTTGTGTATA
GATGGCCTGTATATAGAGGTGTCTAATTCCTTGGCTCTGTATGTATAGGTAATGTGAT
ACTTTACCATTAAAGCACTATTTTCTCCATTCAAGAATTTAGTGATATAGGAAAATG
AGTGGACTTGCGAGACTCAGAAAAACAAAACATAACCTGTCTTGAATTCAAAACAA
ACCATGGGTGTAGGGGGGAACTGATGAAAGTTTATGGGTTTAACTCTAGGTAATTAA
CTAAGACAGTCACGAAACACATTATCAAAATCCTTTCAGGCCCAGAGCTTGTACTGT
ACCCCACTGTGAGACCACATCACAACCCCGGATTGAGCTTTATCCACAACACCTACA
CCATAGTAACGCAAAGTGCACAATGTACTAAAATAAATTCCTATTAGTTTTATGCAA
ACTATGGTATAAAATTATCACCTGCCATACATATTTTGCCATGGCACCAACTTCATAT
AATAAGCCAACGTATAATCAAAGTCCTTACCAGCACCAATCAATGTCCTTGGCACCA
CTGGACACTCACCGTCAAGCTGTTCATCTAAGAGCCAGTCTGTTCTGACCTGAACAG
TTGTGCATTCCACCTTACCACACCCAAGTCTGTGAGCCGGACAAGTGTTTAAATGCA
GTTTTACATCTAACGGTGCAGGTTAAGCCGAGCACTTGAAACTGATCACTCATTAAT
ACCTGTCTCCCTCCATACATGTACACCACATGTACACAGAACTATGTGCTCTGACTTC
AGAATAGCTCTTCCTGTTGGCAAAACACCACAGACATGAAGGGGCCTAGTGTGAAG
CGAGCTCACAGAATGTTGGATGGAACTTCGACTATAATGGAAACACCTGCAAAAGC
TTTGCTAACCCAGCAAACACTCAACACTTACCAAAGACAACAGGGAAGTTAAAGTT
AGCTCGCCAAGAGATGGGCTGGGGAGGTGGGGGTGTAACTCAAAGAAAGCTTTAGC
TAACAAAAACGAATGATGGACAACTTCAGAAATTCCCTAAAAACAGAACCTGAAAG
TGCAGGTGAGGTTTTGTCCTTCAGTAACAAATGCAGACAGATTCCCAACAGGAATAA
AACAGTCTGGGGCTTTGAAACCTGCTAGATGGAAACACGAACTCAAAATGTGGAAC
CAAGGAAAACCAAATACTTAAATGTGTAAGATAATTTATAATAGTAAAAAGTTGCA
AATTGCTGTGACTTGATTTGCCGAAAACATCTGTAAATCCACACTGGCAGTTAGAAG
ACCAGTTCCCACATTAACTCCTCTCTCAGCAGGTAACCGTTTGTGCGCAGAAGTATC
TGAAACATCGCACTACTGCTTATTTTATGGTGTATTGTGCAGAATCTGTACATGCTAT
TACAGACAATACATATTTGTAAACCTGGTCATGCAAAATCAGTGTGTACAAGGGGAT
ATTGTTAAGCCTTATAAAGTGGTACTTTATTATCTTTGTGACGATGCCAATCTCTCCG
AAATATAGCATATCTTAAATGGATATTCTTTATCTGCCAGTTAAAATCATTTTATGTC
ACTGAAAGAAGAGGTTATACAAGGAAAGAAACATGGTCCTTGTGTTGCAGAATTGA
TTTTAAATGAGAGAATTTACAAAACCAAGAAATCCATGGTCATAAAGTTTTAACATT
TTAATCCTACACATTACAGGGCAAACAGATACTGGACCCTATTTCCACATTCCATAA
ATCCAAACTTTAGTTCCCATTTCAAACGTTGCCCTAACCACTAAAACCATCAGTGGT
CTTACAACCTCTGGATTATGGAAATACAGATTTCTGAAGTAAAAGCTACAAAAACAA
CAATGGAAGAAAGCTGAACAAACTTCCCATGAATGAAAATAAAAGTGGAACATCCT
GAAGCTCTAGACACTTCTCTCCCGTGTCTATGGTCAACTTGTCGGTTCAGTGCACTGT
GCGGTCAAATGTAATGGTCCTCATGTGGAACACACGTCTAACTAGTGTCCATTGATT
CCAAGTTAGTGGACGAAGAATCTTTCTGGATACTTTCAAAGATGGCTGCCAGCTCCG
GGTTGGAGCTGATCTGTGACTGGAACTCACTCATGAGAGGGCTCTTCTCTGCCTCTG
GAATGGTGAGCAGTGCAGCTACTGCCCTCATGGCCGAGCGCTTTAACTCGTCCTGCT
TTTCAAACTCCTGCTTTACAGAGTTCGCCTTCACGTAGTTGTACACGTAGCTCGTAG
TGGCTCAACAAGCCGGTCCAACCTCTGTAGTACTGCACTTGGACAAAGGGTAGATAG
TCTCACCAACATTAAAAATGTTAGCATCTTAATATCATAATGGTCCTTCAAACCATCT
TCCACATGATTTAGAAATTCAAAGATATCCAGTCTGTCAAGACAGCTGTCTAGAAGT
GTGTACATACACTCAAAAGCTGCCTTTCTAATGTCCAGGCCGTCATCAACCGTGTGC
TTAAACGGGCCCATCTCTACCTCTCTTATAAGTTCCTTCCTAACTTTTGTCTCATTGTA
AAGATGTGGAAGAACAGAATCCAGAAGGTCCCGTATCAGTGACGGCTTGTTATGGG
CTGCAGAATTGAATGTGACCAAGGCCACTCTTCTTACATTCAAATCTGGGTCTTCCA
ACGTTTTTAGAAAATCACCTATGCAGTTCTTGAGCAGTGGATCTTCTTGACATCTTGA
ATAAACTGACCTACTACAGCCGGTCCCTCTTTAGGGCATGCTCGAGTAAGGGCAGCT
ACACATTTGGCAATGGAATAGTAAGACTGCTTATGAGTAAGAGCTGTGCTCTGAGAG
TAAACTGGACCCGTTAGCATGCGCAGCAAATCCATGTATCCTAGATTGTTTGTTCCA
GTGACAACCAAAGCTTGGAAGAAGTCTAGCATGGCACTAAGAGCTCCTCCCTGCAG
CAGAGGTGACCTTACAAGTCCAATCAGTTCATTGAGAATAGATCCGCTTATCTTTGA
AAGGGAGGAGGGATATACTTTTGCCAGGGTAGTAAGGAAGCTGATAGCCATCTGGG
ACACGTGCATATCACTTTCGCTGATAAGAGGAGGGAGCTCATCCAGAACTGCATCAA
TCATGGCGGCCGTCAAACTGTCACTATAGTTTTTAATGAGAATATCTAGGGCAGAGA
GGGTCCCCAGTTTCAAAGCTCTCTGATTTTTCCTGAGAAATGAAGCAAGGATAGGGA
CTCCCTCTCCCAGCACAGGCCTCAGATCTATCTTCAAAGGTGACCCAGCAATCAGGG
TCAGTGCTTTCACTGTCGTTAGCCGGGTGATTTCATTCTTGAGTCTCTCCAAGAAAAT
CTGAAGTGTATTTGATAAGTCAGGGCCCAAATTGTCTCCAAGATTGCAAATAATCTG
TCCCATACAGGAAATAGCCCTCTCCTTGACTTCCTGATCAATGTCAGCTGCTTTTAAG
CGCTTAATTGTACAAGTGAAGAGATCTTTGATGTAAGGCGTTGCATCGAAGGAGGA
GGGTTGGTCCAGAGGACGGATTACTTTGACAAGCTGCTGAGTGACAAGAAGGGCTT
CTGATGTGATCTTGTAAAATGGGTCACCAACACAAGCCACCACTGGAGGGACCAAA
GCCTGAACATGCGGGTGGAAAACTTGCG-3'.
[0136] This clone is similar to a TATA-binding polypeptide
(TIP120).
[0137] Another IEG nucleic acid clone was designated L048. The
nucleic acid sequence of the L048 clone is as follows:
8 5'-TCGCCGCCCGAAGTCGCGCAGCTTCCCTGGCGAACG (SEQ ID NO:10)
CGGAAGCCCGAAGAGCGCCGTCCTCGGGCCCTGTCGGCGCTCAGGCCCCTTCGCGCG
CCTCCTCGCTCGGCCGGGACGTTGCTGTGGAGGCGTGAGGCGCCGGCGGTCGAGCA
CCTGGAGCGACGGTAGCCCGCGGCCTGCGGTTCTTCTCCTCCCCCGCCGCCCTCCCA
CCCGAGCTGCGGCGGGGCTCGGCCGCCTCGGTGCTTCTGCACGAACAAAGGAGGCC
CCCGCGGCGCCGGCGCAGCTCCATCTGCGGTCCGATCCACCCGGGCCCGCGGCGGCC
GCTAGCCAGCCCTTCCCGGAGGCCTCAGCCCGGCCCACCGCCCGGCGTCGCGCGCCA
GCTCGCTAGTGCATCCGGGCCCCGCAGGCACAAAAATATGGCTCAGGAGACTAACC
AGACCCCAGGGCCCATGCTGTGTAGTACTGGATGTGGCTTTTATGGGAATCCTAGGA
CAAATGGAATGTGTTCTGTTTGCTACAAAGAACATCTTCAGAGACAGCAGAATAGTG
GCAGAATGAGCCCAATGGGGACAGCTAGTGGTTCCAACAGTCCTACCTCAGACTCTG
CGTCTGTACAAAGAGCAGATGCTACTTTAAACAACTGTGAAGGTGCTGCTGGCAGCA
CATCTGAAAAATCAAGAAATGTGCCTGTGGCTGCCTTGCCTGTAACTCAACAAATGA
CAGAAATGAGCATTTCAAGAGAGGACAAAATAACCTCCCCGAAAACAGAGGTGTCA
GAGCCAGTTGTCACTCAGCCCAGTCCATCAGTTTCTCAGCCCAGTTCTTCTCAAAGTG
AAGAAAAAGCTCCTGAGTTGCCCAAACCAAAGAAGAACAGATGTTTTATGTGTAGA
AAGAAAGTTGGCCTTACAGGGTTTGACTGCCGATGTGGAAATTTGTTTTGTGGACTT
CACCGTTACTCTGACAAGCACAACTGTCCTTATGATTACAAAGCAGAAGCTGCAGCA
AAAATCAGAAAAGAAAATCCAGTTGTTGTGGCTGAAAAAATCCAGAGAATATAAAA
TTACTACATGTGAAGAGACTGAAACTTTGTTTTTATTTTAATATATCGTAGGAAAAC
ATTAAAGAGCAGATGCATGGCCATTTTCCTTTGATGTTCTCCAGAGTTTTGCTTTATA
CTTGTCTGTCATATAATTGATATTTTAGGATGTTTGGGTGTTTGTTACAGGCAGAATT
GGATAGATACAGCCCAACAAATGTATATGCCCTCCCCTCAGTAAAATTGGACAAAA
ATATGCACAGCAAATTGAAATACACATATACTAGGAACAAAATTTAGTTCCATGTGC
CAAACTGAATGAAATCTCTGCATGTTTGCAGCATATCTGCCTTTTGGGAATGTAATC
AAGGTATAATCTTTGGCTAGTGTTATGTGCCTGTACTTTAAAAAAATGGTACACCAG
AAAAGGACTGGCAGTCTACTACCATAGTCAAACTTCACCTTAATTTCGACATGCTTT
TGGAAGCAGGAAGAAAGCTACAAAACCAGTATTTGGTGCCATGTGTGAGCCTGGTT
AAATTGGTCTTCTAAAAGCTGTCAATTAGGACATTCTGCGAAAGGTAACATCACAAC
TGGTTCTGAGTAAAACCATCAAGTCAACAGCAGGGTGCCTGAGATAATCTTTGAAGC
TTATTGTGCTGGCCTGCACCAGAAGATATCTGCATTCTCATTACTAAAATTGTAGCAC
AGAACTGCACTAGGATTTGTTTACAAGAAGAAATTAAAACTCTACGTTTGGTTTTCA
CATATAGCAGCTCTGTTAAATAACATGCATCTGAATTTTAAGTTGCAAAGGTATCTG
AGCAGTTAGTTTTTCATGTGCATCTTTTGTTGAATGTTTTGGTTCAAGAAAGAATGTT
TAAAGCTTTTTAAAGACTTCAGTTCTTAATGTAACTGTACCCTTCTGCATGGAAAATC
ATAACCAACATGGCTGCAGTAGACTTCTTTAGTGGTATCCAGCACCACTTGCAGAGG
GCTGCTTTATCATATTGTATTTGGGTGTAGGACTCTAGTGTTCTTGGGTGTATTGCAT
GGGCTGCATTATCTACAGCATTGTACAATAACAACTAGAAAAGGCAGTATACTTCAC
TGATGCTTGTCTGGTAATATCACTTCTGTGTTATAATGGAAGGTTTTTTGTGATGTAT
GAAACTTGTGTTTTTTATATATAAATGAGTATAGTTAGATTAGTGTTGTGGTAATGCC
TGTTTTCATCTGTAAATAGTTAAGTATGTACACAAGGCACTACTTCTGATTTATTGCA
GTGTTCAGTCCTAGTTTTTCTTTATTAAAACATTCAGTTTTGCTTCAATTTTATGTACT
TTAGTTCTAAGTTAGATTTGCAGATGTGTACAGATAGTTCATATTTATGTATTGCAC- A
TAATCATGCTATTCAGCATTGATGCTATATTGTATTATGTAAATAATAAAAGCAGT- G
TACAGAGGGAAAAAAAAACTCGTGC-3'.
[0138] In addition, the L048 clone contains an open reading frame
(ORF) from basepair 414 through basepair 1055. This ORF encodes a
polypeptide of 214 amino acid residues. The amino acid sequence of
the L048 polypeptide is as follows:
9 MAQETNQTPGPMLCSTGCGFYGNPRTNGMCSVCYKEHLQRQ (SEQ ID NO:11)
QNSGRMSPMGTASGSNSPTSDSASVQRADATLNNCEGAAGSTSEKSRNVPVAALPVTQ
QMTEMSISREDKITSPKTEVSEPVVTQPSPSVSQPSSSQSEEKAPELPKPKKNRCFMCRKK
VGLTGFDCRCGNLFCGLHRY SDKHNCPYDYKAEAAAKIRKENPVVVAEKIQRI.
[0139] In addition, the L048 polypeptide was found to be cysteine
rich, having a motif with distant homology to that of polypeptides
with Zn.sup.++-fingers.
[0140] Northern blot analysis using a sequence from the L048 clone
revealed the presence of a 2.5 kb mRNA transcript. In addition,
this analysis revealed that the expression of the L048 mRNA was
strongly upregulated in response to the multiple MECS
treatment.
[0141] Another IEG nucleic acid clone was designated L064. The
following nucleic acid sequence is within the L064 clone:
10 5'-ATTCCAAAAATGCATAGATTACAAAGAAACACC (SEQ ID NO:12)
AGACAAGCTCAAACTCAAGGATATTCTACAAATAAACCAGTACCTTCAAAATGCCAT
GCTACCAGGTACAGACAGGCGAGANACTGTTCCACACTGAGGAAACTAACAAAGTA
TCCATGAAGTCCATAATTGTGGGTCAAATCCAGGACCTGCAAAGGGGATTTGGGGAT
AATTTTCAAAATTTGACTAAGGTCTGCAGAGTAGAGAGACGAGGTCAATGCCAATGT
CCTGATTTTGACAGTAAGTATTTAATATGCAGGAGAACAACCTAACCAAGAGGCTG
CCAACACACTTCCTGGCTGTGGCACAACTAGATTTAAAACCAGCAATTTGTTGGTTC
TTGTTCTCAAATATCAGTTACCTGCAAGCACTCCATCGTGAAAGGATTGAGAGCATG
AGGTGATGTGTTGATGGTGAAAATGAGAACTGACTGAGCACAGGAAAGAGTGGCAT
GATGGGCAGGGAAAGGGGAGACAAAGGTCACAAGAGCATGCAACACTCAGTGAAC
TACAGGACACTCCAAAAGGCACTCTGCTGTCTAGCTTGGATCTGGAGGAGGATCAG
NTATTAATAAGGGCCCTGGAAGGGNCAAAGCTAGCCTCCCAGCTGCTGGCTTCCCAT
CTGCT-3'.
[0142] Another IEG nucleic acid clone was designated L067. The
following nucleic acid sequence is within the L067 clone:
11 5'-GCCACCACCATTGTTAATGGAGGGAGGCTCTCC (SEQ ID NO:13)
CTTGTTATTTCTCAGAAGACTGAATGTCTGTACCAAAAGGCTCATGGCTTTCTCTGGG
CCTTTCCATTTAAGGTTATAGTTTTTTATGTAGTGTTACTAAAATCTAGGCTTGTTACT
AAAGTGGGCTTTGTAGTTATTGGTATCGGTGGATTTTTATGTTACTTGGAGTCCAGAA
CAGGGAGAGCTCACCACAACCTCTCCTTTCCCTGGACCAAACACCCTCTCTGTCCT
GTGAACTCACCTTTTCTTCTCTGTGGGTCACTCCCATTACCACACTGGTGAGCGAGCC
AAATGGATGAGAGACACAAAGACCGTAGTTCTTGAGAGACATTATTTTTTTCAACTT
TGTTTTTTAAGAGATTTTATGTGTTGATTTGTTTTGGTTTGGTTTAAAGGGATTCATA
GCTAACTTGGATTTTTGTTACCTCAGCTCTGGGAGAGGATTTTTGCTGAATGACTATT
AATTACCTGAGCATTGTTGCTCTGAGGTCATGGCATGCTAGCCTATGTCTGTTACAGT
CTCAGGCTGCCCTTGTTTCCTCGTTCCTGTGCTATTGTGCTACACGCTCAAGGGGCCT
TGACTCTGCTTACACACATTAGGGGCAGTGTGAGTAAATGTGCAGTGTCCACACTTG
AGGACATGAATGTCTGCACTGTCACTTTGCTCTGGGTGTGAAGTCCCTGGTCCCCTTG
CTCCTGTAGCTTTCTTTTGATCGACTACTGGAACTCAACCCTGTGTACAAGAGCAGC
ACTGCCTCTGGTGGGTGGTGTTTGCAGCCAGGATTAGATGCCAGTCCTCGGGTTCCC
TGGCCTTGTTGGAAAGGTGTGCTTCCTTGAGGTCTGAGAATGGAAGGCTCTGCCTCA
CTCTAGCTAGGAGGCGCAATGGGAAAGTATGAGTTCAGGGCGTCAGGGCAGTGGCT
CCTGAAGAGCCAGCTGTGGACAGAGGGAGTGAGGCTTTATTTAAAGTGACAGGAAG
AAACATGGCGTTTTGGTATATTGGGAGCAATGCCAAGATTCCCTCCTGCCCTACATA
GGTCACAGACACCTTCCCAACCATCCCCTCCTCCACTTCCATAAATGAAGACAGCCC
TGATGACCCTCACCCCTTTTGCATAGGTCACTGGATCCCACTGTCCTTCCTCGGTGCT
TACACACTTTACAGACCCTTTAGGCGAGCCCTTGCATAGAGCGTTATCTCAGTGCTC
CATTCCAGTCCTGACTCCCTGTGGCCATTGAGACTTTGGATTTAAGAACTCACATTGC
TAGGGAGAGGGGCTTTGCTGGGAAAGGTGACTCCTCTGTAACCTAGCCTCTTGTGCT
CCTCCATGACAGAAATGCTGGGTGGAGTTTTACATTTGCCAATGGCCAGCTTGTGAA
TATCTTCATATACACTTTCTATTCATGTTACTGTAGTTTCTGTTTTGAAATAAAACTTC
TGAATGT-3'.
[0143] This clone is similar to a glucose transporter type III
polypeptide.
[0144] Another IEG nucleic acid clone was designated L076. The
following two nucleic acid sequences are within the L076 clone:
12 5'-CATATAAATGTACTTTATTGTTTTAAACAGAACG (SEQ ID NO:14)
AAAGAAGAGGCAGAAAACATTTGCATGTAAGTCCTAGCTTATAAATGTAGTTTTTAG
TGGTGGCATCTCTAACACGTCGTTCAGGGACTGTTTCCTTTTGCCTCCTTGTACTGTG
AGCACTGACACTTGAGAAAAGCACATCTGGCGGACATATGTCTCCAGAACTGGAAG
AACTTGGAGAGCAAACATTTTTCTTAATTCCTCTAAGTAATCTTTAGTAAAACAAAA
GATGATCTTTGGCATAGATTCATACTTAAAGGCATTGATATGCATTTATATCAGGTA
AGCAACTATACAGATCTGCTGAGAGCTTTCAAAAGAATCTGTTATCAGCTGAAAGGA
AATAGGGGAAGCCTGAGTATTCAGGGTCAACTTAAGATTTGCAAGTTCAGTGTTGGG
GTCAACATACTAGATGTGGGAAGAACATCCAGGCAAGGTCTTAGTCCTGTATTCACC
TGGTTCTTGATTTCTGGAAGAAGCATCCATGCGCTAGGAAATGCTTATACAGCCGAG
GTAAATGCAAAAATGAGTAAAGTCACTTTTTCACTAACTTTGCCCAATAGGRAACAT
GCCTTTCTGATAAGTAGATACCATACTCTTTATTCTTGAATACTTTATATTGAGAGAA
GGTTGTAGTTGGTTAAAAGCAACTGGGAACTATAACTTCCTACTGATTTTTCCCTAGC
AGCACCAGAATTATATTCTGCAAATGCTATTCTCCCTTACATAGGAAATATCCTTCA
GACAAAATTGCCTTTCCATTCAGTCTCTTAAGAGYTTAATTTTGAATGGACTTTTCAA
AGTTACAAGCAAAGTCAAGTGTGGTGGTAGGAGCTAAGAGGCTGACACAAGTAGAT
GACTTGAATCCAGAAGTTCAAGACTAGCCTGGACAACATAGAGAGACCCAGTCTCA AAATT-3'
and 5'-GGCGGGGATCTCTCGGCTGGTAAGAA- GGGG (SEQ ID NO:15)
CAGTGGTACCANGCGGGCACTTATTCAGTGTGCCAAGG- ATATCGCCAAGGCCTCTGA
TGAGGTGACGAGGTTGGCCAAGGAGGTTGCCAAGCAGT- GCACAGATAANGCGGNTT
AGAACCAATCTCTTACAGGTCTGTGAGCGAATCCCAACT- ATAAGCACCCAGCTCAAA
ATCCTGTCCACAGTGAAGGCCACCATGCTGGGCCGGACC- AACATCAGTGACGAGGA
GTCTGAGCAGGCCACAGAGATGCTGGTTCATAATGCCCAG- AACCTCATGCAGTCTGT
GNAAGAGACTGTGCGAGAGGCCGAAGCTGCTTCAATCAAG- ATTCGANCAGACGCCG
GATTTACTCTGCGCTGGGTCAGAAAGACTCCCTGGTACCAG- TAGGCACCTGGTCAGA
CCTGGCTGGTACACAGACCTCTGCTAATGANGANGTGACCA- TCTTGAGCTTCAGAAG
CCATTCAGAGTTGCCAAGGGGTGGNAAATCAATCCCTGGTT- TCACACACCAAGAAA
GGGAATGGGGCCTCCTTCACATTAGAATAAACATTTATACTC- TTGTCATGGGACACT
TTGAAAGTGTCTCTCCTACAAAACCCCTGGTACCTTTCAGGN- TTACTCCNGGTNGCA
ANNTCCTCCCCCAAGGGGAATTTTTTACCAATAAAAGGCTCA- AGGAATTAANGGCG
NTTGAAAACCAACNTNATCCAANGGGAAANGCCCCCNTGGCCT- TCTGGCCCCCTTGG
GGGNACAATTTTTCNTCCCNCTGGGTGTTTTAAATGGGGTTTC- AACCTTGGGGCTGG
NCCTTTTTCCNCCCCCCCTTTTAAGGGGCTTCCTCCGAAGGAA- CCTNAGAAAACTTN
AAGGGCCAAAGNTCCANTTTACNAATAACTGGG-3'.
[0145] This clone is similar to vinculin.
[0146] Another IEG nucleic acid clone was designated L082. The
following two nucleic acid sequences are within the L082 clone:
13 5'-TTTTTTTTTTTTTTTTTTCCTCCCTTAAAAGAT (SEQ ID NO:16)
AAACTAATAAACTCTTCAATGGTCTTTTCAGTATAGTTCTTATGTAGTTTAACATAGC
TTATAAATTGAGTTTAACAATAAACTCAAGAAGATAATTTTATAAACCCTGTTTTCC
AATCTGTCATTTACTTAAATTATTTTGGTTGTTTTCCCTTTTTTTCCTTCTTTCTCACCC
CCTCCCTCTCCATGAAGATTCAGGTGCTTAACATATCATTTTTTTCCCTGCTGGAATT
TTAGCATTGATATGAACCATGGACAAGTATATTCTGCTGCCACAAAGACTGTAAAGT
GCTTCATTTCAACAGCTGAGGCAAGCCAAGTGATCATTAATAAAGCTTTTCTTGCTTC
CTTCAGTGGTGTTGGTAGTAAAATGGTAGGTAAAAGTTAGGCTGCAAGTTCAATAAA
TGAGATTTACCTATCATTCCACCCTTGTGTATTCATTCACCTATCCTGGTTCAAGCAG
TTTGAGTCAACTAGGCATTTAAAGGCATTGTGTTTATTACTTTATGGTTCCAACTTTA
CATACTTGTCAGGGATGAAGTCTGATAGGTTAAGGACAGTAGAAATTTCTGTGCAAC
AAGCAGCAAC-3' and 5'-TTTTTTTTTTTTTTTTGGTTACAAAAGT (SEQ ID NO:17)
ATTTATTTTATAAAACTTGTATTTAAAATAGAGCTTATCTGTCTACTCACAAATCCTA
ATTTAAAACATAACACATTATCCTTAGCTAATCTGATGTTAACCTTTACAATCAACAC
TCATTTTTGTAATTTTATTAAGAACCTGTACTAAATGAAGTTTTTAATCAGAAAACAT
TCCCTTTTATCTTAAAAGTGCTTCTTAAATGAAGGCACCAACAAGAACTACTTTCAG
ATGGTACAGAATTTCTTATTTCTTGAAGACTCTGTGGTTGACCACTTCTTCATTAGTT
ACCTGCAGCAAGACACCTTCCTGCCAAAGGAAAAAAAAAAGTATCTGAAGAAGTTT
ATCATGTTTGTCCAAAGAACCTAAGTAACTTCAGTGGTGGTTTTAGGATTAAAGCAG
ACTCACTGATGTGTATACGCCCTGAATATCACATTTCTGGAAAGGCAGTAAAGCCTA
GAAATCAGAAGGCGGGCGGTTTTAAAGAAATTTCAATAGCCAACCTACAACANTTT
AGGGCAAAGATAATGGGCAAAAANTNC-3'.
[0147] This clone is potentially similar to a nRNP polypeptide
A2/B1.
[0148] Another IEG nucleic acid clone was designated L094. The
following two nucleic acid sequences are within the L094 clone:
14 5'-ACGATATMTAYWGARRTWYAWCTSTTHAC (SEQ ID NO:18)
TGAATMWHATGCACAAATATTAACTAGTRRTTTATTAAACAGATATSATTTAGAACA
AGACTTAAWKAAATACAAATCCTTAGGTACGRTTTAATATCATGTTCADGATGTTTG
AAGAGTTTAAAAAGAATCACTGATTAAGKKAAGCATCCBCACTTTTCTTTGAGAABC
CAAACCTTTTAGGNAAADACCCCATTCCAAATTTTGTCCCCHATTTCAGRCCKKCAG
AAAGTCTCTAACATSAAGAGTCCTCAACGGGGNGTAACTCAVAWCTCCTATCAAGT
GCAGTAACCTAGCTCTCCCGGDGGCCATGGCGT-3' and 5'-AAACT (SEQ ID NO:19)
AAACAGTGTTTTGTTAATTCTTCTGCATTCGGACTATTGCAG- GCATTAGAGCATCCAG
AGCTACGAAGGGCTGGCTGCAGCAGCACCGCCCTTTGTAAG- CCAGCAGACCAGCCT
TAACTGTGGGCTTGACTCCTGTGAGCTGGCCTCAGTGTGACT- CAGAAATGTTTGATT
AGCAGATGAGAGAGCGAGGACACACCACGAGGGCTGCGTTCT- CTTCCTCCAGCGCT
GTGCAGGACAGTTTCTTCTCACCCTAGCCTTTTTAAATGCACC- AGAAGTACAGACAG
TTGCACTACACAAACCCTTTGAACACTTGTAGAAATCAGTCCA- CCGTAGATTAGACA
GAATCACCTTCCAATCCTTTGACTTCTTTTCCTTTCATTTGAA- CAATTGTATAATAATT
GATTATTGTCAAATTTTTGTCTGTGGTAGTATCGCTTTAAT- TTATCTTAGTACATCAA
CGTTTTGATTTAAAAAAGAATTAAAACAACAAAAAAAGTC- ACTTAGAAGCCATGAA
CTTTTTTTTTTNGATNGGGAAATTTTCTTGTTTNGAAAATT- ATCATTGGGGTTCCTCC
GGAAANCTTGTAAGATTGGNTTATAAGGTACCTGGGANGT- TCANAACNGGTGGNTA
TACCCTTTTTTAAGGGAAATTAATGATTTNGAGTTTTTGGG- CCAACTNCGGGANTGG
CAGGGAAACCANNCNGGGGNGGGGTTTAAATTNTGTGAGGG- TTTTTTGGGCCTNAA
TTTTTTGCATAATTTTCACCTNGNAACCTTTNAANNCTNGGA- AAAAAAAAAAAACNT-3'.
[0149] Northern blot analysis using a sequence from the L094 clone
revealed that the expression of the L094 mRNA was upregulated in
response to the multiple MECS treatment. Specifically, L094 mRNA
expression was induced 7.3 fold by the multiple MECS treatment as
determined from Northern blot data using total RNA from rat
hippocampus (Table I). In addition, developmental studies revealed
that the transcriptional expression level of L094 was upregulated
between day E15 and E18, and downregulated at day 0. The expression
then increases again during post natal development.
[0150] Another IEG nucleic acid clone was designated L097. The
5'-end of the clone obtained from the first library screen was used
to design an antisense primer. Using PCR, L097 DNA was amplified
and inserted into the pCR2.1 vector. The L097 clone is about 4.4 kb
in length. Sequence analysis of the first 4060 bases from the
3'-end revealed the presence of a coding region of at least 2351
bp. In addition, RT-PCR analysis revealed that the L097 clone was
missing an adenosine at position 1166 from the 5'-end. The lack of
this base results in a frame shift in the coding sequence. Further,
the sequence at position 1358 was ambiguous. However, any base
substitution at this particular position will not alter the encoded
amino acid residue. Specifically, a serine residue will be encoded
by the codon containing nucleic acid position 1358 regardless of
the base at position 1358. The following nucleic acid sequence is
within the L097 clone:
15 5'-TGCAGCCGCCCTTGGAACTGCATGTCAGGAAGCATCCCTTTGTGTA (SEQ ID NO:20)
TGTCTGTGCTATATGTCTCAAGAAATTTGTCAGCTCAATCAGGCTGCGCTCCCATA- TC
CGAGAGGTGCATGGGGCGGCCCAGGAGACCTTGGTTTTTACTAGCTCCATCAACC- AG
AGTTTCTGCCTCCTGGAGCCTGGTGGGGATATCCAGCAGGAAGCCTTGGGAAACC- AG
CTATCACTGACAGCTGAGGAATTTGTGTGTCCAGAAATTGATGTACGTAAGGGGG- AG
GTTTGTCCTGGGGAAGCTCAGCCTGAGGTGGGGCTGAGGGAGTTGGAGGCCCCTG- G
AGAAGCATGTGCCCCAGCCGTGCCCTTGGCCAACCCCCAGAGTGTCAGTGTTTCCC- T
GTCCCCCTGCAAACTGGAAACCACTGTGGTCAATTCCGACCTCAACTCTCTTGGAG- T
GGTTTCAGATGATTTTTTACTGAAAACTGATACCTCTTCTGCTGAGCCTCATGCTG- CT
GCTGAGCTAACCTCAGACACACAGCATCGAGGCTCAGCCCAGACTCAGGGTGAAG- A
AGTCACACTGCTGCTGGCCAAGGCCAAAAGTACTGGACCAGACTCAGAGAGTCCTC
CAAGTGGAGGGCAGAATGTGGGTGCTCTGCCAGCCAGTGAATCTGACTCTAACAGG
TGTCTCAGGGCAAACCCAGCAGAGACCTCAGACCTCCTTCCTACAGTGGCTGATGGA
GGAGACCTCGGTGTGTGCCAGCCTGACTCTTGCACGTCGTCCTCTGAGCACCACCCT
GGCAGCACAGCATTCATGAAGGTCCTAGACAGTCTCCAGAAGAAGCAGATGAACAC
CAGTCTTTGCGAGCGGATCCGGAAGGTTTATGGAGACCTGGAGTGTGAATACTGTGG
CAAACTTTTTTGGTACCAAGTGCATTTTGACATGCATGTCCGCACCCACACCCGGGA
ACATCTGTATTATTGCTCCCAGTGTCACTACTCTTCCATCACCAAAAACTGCCTTAA- A
CGCCATGTAATTCAGAAACACAGTAACATCTTGCTGAAGTGTCCCACTGACGGCTG- T
GACTACTCGACTCCAGATAAATATAAGCTACAGGCCCACCTTAAAGTTCACACAGA- G
CTGGACAAAAGGAGTTATTCTTGTCCTGTATGTGAAAAATCTTTTTCAGAAGACCG- A
TTGATAAAGTCACATATCAAGACTAATCATCCAGAGGTCTCCATGAATACCATTTC- T
GAGGTTCTTGGGAGAAGAGTCCAGCTCAAAGGGCTAATTGGAAAGCGAGCCATGAA
GTGTCCGTATTGCGATTTCTATTTCATGAAGAATGGCTCAGACCTTCAGCGGCACAT
CTCNGCTCACGAGGGTGTGAAGCCCTTCAAATGTTCTTTGTGTGAGTATGCAACTCG
TAGCAAGAGCAACCTCAAAGCTCATATGAATCGTCACAGCACTGAGAAGACTCACC
TCTGTGACATGTGTGGCAAGAAATTCAAATCCAAAGGGACATTAAAGAGTCATAAG
CTCCTTCACACATCTGATGGGAAGCAATTCAAGTGCACGGTGTGTGACTACACAGCT
GCCCAGAAACCACAGCTGCTGCGACACATGGAGCAGGATGCCTCCTTCAAGCCTTTC
CGCTGCGCTCACTGTCATTATTCATGTAACATCTCTGGATCTCTGAAACGGCACTAC- A
ACAGGAAGCACCCCAACGAGGAGTATGCCAACGTGGGCAGCGGGGAGCTTGCAGCT
GAAGCCCTCATCCAACAAGGTGGTCTGAAGTGTCCTGTTTGCAGCTTTGTGTATGGA
ACCAAATGGGAGTTCAACAGACACTTGAAGAACAAGCATGGCTTGAAGCCAGCGAC
AGAGACTCCCGAGGAGCCCTCCACCCAGTATCTCTACATCACCGAGGCTGAAGATGT
TCAGGGGACACAAGCAGCTGTAGCTGCACTTCAGGACCTGCGATATACCTCCGAGA
GTGGTGATCGACTTGACCCCACAGCTGTGAATATCCTGCAGCAGATCATTGAACTGG
GTTCAGAGACTCACGATGCTGCTGCCGTGGCCTCCGTGGTTGCCATGGCGCCTGGGA
CAGTGACTGTTGTAAAGCAGGTCACCGATGAGGAACCCAATTCCAACCATACAGTC
ATGATCCAGGAGACTCTGCAGCAGGCCTCTGTGGAGTTGGCCGAGCAGCACCATCTG
GTGGTGTCCTCTGATGACGTGGAGGGCATTGAGACAGTGACAGTGTACACACAGGG
TGGGGAGGCCTCAGAGTTCATCGTGTACGTGCAAGAGGCTGTCCAGCCCATGGAGG
AGCAGGTCGGGGAGCAGCCAGCCACAGAACTCTAGAGAATCCCTGCCTCCTTTGGC
AGCCAGCCTTTGTGGGCCTGAAGACCTCCTAACCCACCAGGTCCATCCCTGGCTCTT
CTTGCCCACTGGCCCCAGATAAATTTCTCCATAACTGTCCTCTGTGTGGTCAAAGCC- A
GGAGAGTATCATGAAGAGAGAGAGAGAGAGAGACTAGTCTCCGAGTTTTTTTTTT-- 3'.
[0151] In addition, the following amino acid sequence is within the
L097 polypeptide:
16 QPPLELHVRKHPFVYVCAICLKKFVSSIRLRSHIREVHGAAQETLV (SEQ ID NO:21)
FTSSINQSFCLLEPGGDIQQEALGNQLSLTAEEFVCPEIDVRKGEVCPGEAQPEVGLRE- LE
APGEACAPAVPLANPQSVSVSLSPCKLETTVVNSDLNSLGVVSDDFLLKTDTSSA- EPHAA
AELTSDTQHRGSAQTQGEEVTLLLAKAKSTGPDSESPPSGGQNVGALPASES- DSNRCLR
ANPAETSDLLPTVADGGDLGVCQPDSCTSSSEHHPGSTAFMKVLDSLQKK- QMNTSLCER
IRKVYGDLECEYCGKLFWYQVHFDMHVRTHTREHLYYCSQCHYSSITK- NCLKRHVIQK
HSNILLKCPTDGCDYSTPDKYKLQAHLKVHTELDKRSYSCPVCEKSF- SEDRLIKSHIKTN
HPEVSMNTISEVLGRRVQLKGLIGKRAMKCPYCDFYFMKNGSDL- QRHISAHEGVKPFKC
SLCEYATRSKSNLKAHMNRHSTEKTHLCDMCGKKFKSKGTLK- SHKLLHTSDGKQFKCT
VCDYTAAQKPQLLRHMEQDASFKPFRCAHCHYSCNISGSLK- RHYNRKHPNEEYANVGS
GELAAEALIQQGGLKCPVCSFVYGTKWEFNRHLKNKHGLK- PATETPEEPSTQYLYITEA
EDVQGTQAAVAALQDLRYTSESGDRLDPTAVNILQQII- ELGSETHDAAAVASVVAMAPG
TVTVVKQVTDEEPNSNHTVMIQETLQQASVELAEQH- HLVVSSDDVEGIETVTVYTQGGE
ASEFIVYVQEAVQPMEEQVGEQPAT EL.
[0152] Using tblast2x algorithms, nine Zn.sup.++-fingers were
identified by homology to motifs of Zn.sup.++-finger containing
polypeptides (accession # PIR2:A32368, S03677, A29634, S06571, and
A60392). The presence of the multiple Zn.sup.++-finger domains
suggests that the L097 clone is a transcription factor, however,
the size of the encoded polypeptide is in excess of 700 amino
acids.
[0153] Northern blot analysis using a sequence from the L097 clone
indicated that the L097 mRNA transcript is rather rare. In
addition, this analysis revealed that the expression of the L097
mRNA was very weakly upregulated in response to the multiple MECS
treatment.
[0154] Another IEG nucleic acid clone was designated L099. The
following four nucleic acid sequences are within the L099
clone:
17 5'-TGGATCTACTTGTTAATGGTTTCATGGAAGC (SEQ ID NO:22)
AATCAGCAATATGTGATATGAACTGCTGCATTACTTATTATACTCGTGGAACTGAGA
TATTTARMSRSMGCTTWWYTTTTTTTTTTYTTAGTGTAAAATACTTAAGCGTTTCCAC
TATTGGAAGAAAAGCATATATGGGTATTTTGTATTGTAACTTGTTTAAAAGGACAGT
CTTTTTTAAYCTTCCCACTTAAATGCTTTAAAATATGTAATACAATTTGAAGCTTGT
TTAAAAATAGAATTAAATGTCTTAWATAGKGCTACKGTTTTGGAATTAGAAAGTGAT
CAAATACAAAACATTTTAAAATTAAGCCCAGAAAACAAAATAGTGTTTAAAGTTAG
TTTAGTATAAAAGAAATTTATAAGATTTTTTCTTCAATATAAGATACCTCACTTGAAA
ATAAAGAAAGCACAGCACATTAAAGTAATTCTCATGAGAACACCCCATTAGAATAA
TTGCTAAATCTAGGACACCTTTTGAGTTGTGAGTTTGTGATACATGTAGTCACCATTA
GCTTTTCTGCTGGAAGGACTTCCCGTAGTAATTTTAAGGCAGTGTAATAGTTCAATTA
CCCCACAGTTTCTAACCTGGGAAGGCAGTATGTGAATGGTCCCTTCTGCAACTACGG
AAACACATTAGCTACATTGAGCATAACTCGATTGATAATTTTGCCAGTGCATATAGT
TTTATGATTAAAATTGCTGTGGTTGGTTGCATTACACGACACACAAAACTGTCCTCTA
CCTCACATGAAATAAATATTTTATATGGTTTTACTAAAAAAATGACTCATCTATCTGG
TTACTTAGTTTACAAATTTTGGATTATATTTATTGAAACATGACATACTGTGCTCTTA
GCTTATACCTCAATCGTATTTTGTGCTGTTTGCCATTTTCATGCCTTGTATATAACTTG
TATAGATTGGATGATATTCCCAATAAACACTTTTAATKCCAAWRAAAAAAAAAAAA AAAAA-3';
5'-TAATGTTTATGATACAAAGCTACTCACTCTG (SEQ ID NO:23)
GAGCCTTCTCATTACAGAATCTCTTGACTTTTATACACCCAG- CCTGTTGTTACTTTGT
TCAGGTTGCAGAATGAGTTTCCTCTGGTTTCCTCCTAGAGG- AGTTTTCCTGATGAAAT
GCTAGTAGCACCTCCCCGACATACAGCGGGTGGGTGGGGC- ACACTTTGCTGTGCTCT
GATGGTACACACAAGAAGCAGTTGTAATTTGTCTTTCTGT- TTAAGAGTGACCATAGC
TAGATATGTGTGTGTGACTTCAGAAAATTAAAATGCTTTC- CGAACTTTTCCTGTTAAT
AGAGGTGTGAAGTACTCATTCATGTGCATGAGGAAAGTG- GATTCCACGGACGCACA
CCGCTTCCTATGTAACTCACAATGCTCTGTACAGTTTTTA- TATGTAGTCTTACAAAGG
TCTTATGAAATTTATATAATGGATTTTTTCTTTTAAATT- ATAAAATACTAAATATCTT
AAAGATTGTTTTGGACTTTTGTATGTTTAAATGTTATC- TTAAAACTTGCACAAATGGA
CCATGATGACTCTTTGATCTTAAAATCAGGAATTTAC- AGTCAGCTAAGAAAAATGTG
GATAGGTTAATAATCCACAGTGGGAGTATCTGCTAGG- AGCAGGAATTGTAGATGAC
ATGAATTCCGTGATTTGAGGAAGGGCAGCCTCTGCACT- TTTCTTTGTTTTTGTTTTTT
GCACATGAAGTCTGACATTTTTACCATCGAATTTCAC- ATTACTAGATGGTTGGCTTGG
GATTTACCTAGGGGAAATTCTTAGCAACTTTGTACT- TTGTTGTTTTTGTTCTGTTTGGT
CTCCAGCTTGCAGAGACCCTCTTGCCTCTGTCTC- CCAAGTGTTGGGTTGGCAGGATG
AGCCCCACCACCGCTGGCCCTGTGCAGTTCTTTT- GGGATGTCCCTGAAAGCAGCTGT
GGCATTATCTTCTGTTTCATGTGTCCCGAGCTGT- CTCATGGTACTACATGCAGTGACC
TGAGATCTGCGTTAAGGAATAACTTAGGAGAAA- ACGGCTGTCACTGTCCTCCCCGCT
GTGAGACACCAGAGTTATCACACCTGTTATGGT- CATACTTTGTGTTATGATACTGAT
GTCTAAGGCAATTTTTCTACTTTCCAAAAGGGA- GTTTGTTTCCTAAATATATTGTGAC
CTAAATGTGGTTTTATTCTGCTATGTTCTATA- ATTTATGTATTGACTTTTGTAACCTCC
TTGGGAGAAACATGTTAAGTGGCACAGGGA- CCATATATGTCATTTTATTTAGCTCTG
GAGAAGGAAACCACAGGCGTTTGTAAAATA- GCATTAGCTTAGATGTCAGTTCATTGT
GCTTGGCTGTGTGGGAGGCAGACTCAAGGA- CTTGCACCATTTATTTTTCTGACAGAA
GTGTTCTGCTTATGTGCTGCTTAGTAAGTG- TGATTTTTCTAGTCTTGATGAAACTTGC
CTCGTGACATTGTTGAGCGTAGTCTTCAC- TTTCCAGAAGATGAAATGATGTGCCATC
ATTTTCTGTCTAAACTTCCTTTAAAGTAA- TTTTTAATCAGCTGTAAATATCATATCTC
CTACTGTTGAAAGTAACTTTAATTTACA- TTGCACCATATAGCTTGAAAACCAACTTTG
AAATTCTGTACTCCTCCACAAGTGACC- TCCGCTAAAATACCCATAGGAAGCTTACTT
TGTGCATGCNTGCTTTGTGTGCCGGTT- GCCGTCCTAANGGTTGCTTTGGG-3';
5'-TTTTTTTTTTTTTTTTTTAGTGTAAAA- TACTTAAGCGTTTCCACTA (SEQ ID NO:24)
TTGGAAGAAAAGCATATATGGGTATTTTGTATTGTAACTTGTTTAAAAGGACAGTCT
TTTTTAATCTTCCCACTTAAATGCTTTTAAAATATGTAATACAATTTGAAGCTTGTTT
AAAAATAGAATTAAATGTCTTATATAGTGCTACTGTTTTGGAATTAGAAAGTGATCA
AATACAAAACATTTTAAAATTAAGCCCAGAAAACAAAATAGTGTTTAAAGTTAGTTT
AGTATAAAAGAAATTTATGAGATTTTTTCTTCAATATAAGATACCTCACTTGAAAAT
AAAGAAAGCACAGCACATTAAAGTAATTCTCATGAGAACACCCCATTAGAATAATT
GCTAAATCTAGGACACCTTTTGAGTTGTGAAGTTTGTGATACATGTAGTCACCATTA
GCTTTTCTGCTGGAAGGACTTCCCGTAGTAATTTTAAAGNAGTGTAATAAGTTCAAT
TANCCCACAAGTTTCTAANCTGGGAAAGNAANTATGGTGAATGGNCCCTTCTGCAAC
TACGGGAACACA-3'; and 5'-TTTTTTTTTTTTTTTTTTTTTGGCATTAA (SEQ ID
NO:25) AAGTGTTTATTGGGAATATCATCCAATCTATACAAGTTATATACAAG- GCATGAAAAT
GGCAAACAGCACAAAATACGATTGAGGTATAAGCTAAGAGCACAGTA- TGTCATGTT
TCAATAAATATAATCCAAAATTTGTAAACTAAGTAACCAGATAGATGA- GTCATTTTT
TTAGTAAAACCATATAAAATATTTATTTCATGTGAGGTAGAGGACAGT- TTTGTGTGT
CGTGTAATGCAACCAACCACAGCAATTTTAATCATAAAACTATATGCA- CTGGCAAAA
TTATCAATCGAGTTATGCTCAATGTAGCTAATGTGTTTCCGTAGTTGC- AGAAGGGAC
CATTCACATACTGCCTTCCCAGGTTAGAAACTGTGGGGTAATTGAACT- ATTACACTG
CCTTAAAATTACTACGGGAAGTCCTTCCAGCAGAAAAGCTAATGGTGA- CTACATGTA
TCACAAACTCACAACTCAAAAGGTGTCCTAGATTTAGCAATTATTCTA- ATGGGGTGT
TCTCATGAGAATTACTTTAATGTGCTGTGCTTTCTTTATTTCAAGTGA- GGTATCTTAT
ATTGAAGAAAAAATCCATAA-3'.
[0155] This clone is similar to sno I.
[0156] Another IEG nucleic acid clone was designated L100. The L100
clone is 2924 bp in length and has a nucleic acid sequence as
follows:
18 5'-TGCGGCCGCCGGGGCCGGG (SEQ ID NO:26)
GCTGAGCCAGTCTCTCCCGCCGCCGCCGGACGCGCAGACCTGGGCAGGCTGCACCG
ACGGCCGCCTGGCCGAGCGCACTGCAGGTCGCTGCGCGCGCTGCGACCCCGGGGCC
CGGACGCGAGTGGCTGCGGTGTCCTGGGCGAGCACTGCTAGTTTAGGCCGTCTGTCC
TCAGCTGCTTGGAACCCCTACATCCCACCATGGCTGGGATACAGAAGAGGAAGTTTG
ACCAGCTGGAAGAGGACGACTGCAGCTCCTCCTCCTTGTCCTCTGGCGATCTCTCTC
CCTCTCCTCCCAGCTCTTCTGCCTCCCCTGCCTGGACCTCTGAGGAGGAGGGACTGG
GTGATCAGCCACCCCAGCCTGATCAGGACTCCAGTGGCATCCAGAGTTTAACGCCCC
CATCCATCCTGAAGCGGGCTCCTCGGGAGCGTCCGGGTCACGTGGCCTTCGATGGCA
TCACTGTCTACTATTTCCCGCGGTGCCAGGGATTCACCAGTGTGCCCAGCCATGGTG
GCTGTACCCTGGGCATGGCTTCTCGTCATAGCACCTGCCGCCTCTTCTCCTTAGCCGA
GTTTAAACAGGAGCAGTTCCGGGCTCGGCGTGAGAAGCTCCGTCGGCGTTTAAAGG
AGGAGAAGCTAGAGATGCTGAAATGGAAGCTTTCAGTGTCCGGAGTTCCGGAGGCA
GGGGCAGACGTGCCGCTCACAGTGGACGCCATCGATGACGCTTCTGTAGAGGAGGA
CTTGGCAGTGGCCGTGGCAGGTGGCCGCCTGGAGGAAGCGAATTTCCTACAGCCCTA
TCCACCTCGGCAGCGACGGGCCCTACTTCGCGCTTCCGGTGTTCGAAGGATTGACCG
AGAGGAGAAGCACGAGCTGCAGGCGCTACGCCAATCCCGGGAGGATTGTGGTTGTC
ACTGTGATGGCGTCTGTGACCCTGAGACCTGCAGTTGCATCCTGGCGGGCATTAAAT
GCCAGATGGATCACACGTCCTTCCCCTGTGGCTGCTGCAGCGAGGGCTGTGAGAACC
CCCATGGTCGAGTGGAATTCAATCAGGCGAGAGTTCAGACACACTTCATCCACACGC
TCACCCGCCTGCAGATGGAGCAGGGTGCGGAGAGTTTGGGGGACCCGGAGTCCCCC
ATGGAGGACGTTCCTGTCGAACAAACCGTGGTTTCCCCCTTTCCTCCTTCCAAACCCA
CTATGAGCAATGACCTGGGGGACAGCAGCTGTGGCAGCGACATGACAGACTCTTCC
ACGACCTACTCCTCTGGCGGCAGTGGCAGCCGCAGCGAGGCTCCGAACCATCTTGCC
CACCCCAGCCTGCCAGGTTCCAGCTTCCGGTCTGGCATAGATGAAGACAGCCTGGAA
CAGATCCTGAATTTCAGTGACTCTGACCTCGGTATTGAGGAAGAAGAGGAGGAGGG
AGGGAGTGTGGGCAACTTGGATAACCTCAGCTGTTTTCATTTGGCTGACATCTTTGG
TACCGGTGACCCCGGCAGCCTGGCTAGCTGGACACACAGCCAGTTTGGCTCTAGCCT
TCCATCGGGCATCCTAGATGAGAATGCCAACCTGGACGCCAGCTGCTTCCTAAGCAG
CGGACTCGAAGGGTTGAGAGAAGGTAGCCTCCCCAGCAGTTCTGGGTCCCCTGAGG
GGGAAGCCGCCCAGAGCAGCTCCTTGGACCTCAGTTTATCCTCCTGTGACTCCTTTG
AGCTTCTCCAATCTCTGCCAGATTATAGTCTGGGGCCTCACTATACTTCCCGAAGGGT
ATCTGGCAGCCTGGACAGCCTTGAGACCTTCCACCCTTCGCCCAGCTTCTCTCCACCG
AGGGATGCCAGCTTCCTGGATTCTCTCATAGGCCTGTCTGAGCCGGTTACAGATGTC
CTGGCGCCCCTTCTGGAGAGCCAGTTTGAGGACACTGCTGTGGTGCCTTTGGACCCT
GTGCCTGTGTAAGGATTGAGATGACTTTTTCCTGCCCTGAGACCCTGTTGCTGCTTTT
TATGTGATCTTGGTGTCCCCCAAGGTCTGTGTATGTAACGGTCTCCCGTGGGCTGGTT
CTGCCCCCGTGCCATGTGGGCAATCCTCTATTTTTACAGTAACACTCTAGATTTATTT
ATTTTTTTATGTTTTTCTGTACTGAAGGGAGGGTGGGAAGGGTATCCCTCTTTCAATG
CCTGGCCTCTATGTCCAAACAGAGGTCTCCCACCTCCTACTGTATGCCTGGAGGAGG
AAGGGGCGGGGTTCACATCCCCTCTTTCTGTACTGTAAAATGCTCCTTGGTCCAAAG
ACAGCTGAAAAGCAGGCCTTAGGGTTTCCTGTGGACCGTGGGAGCTAGGTCTTCTGG
ACTCTGAAGATGTAATTTATTTCTGTAATTTATTTGGGGACTGAGACAGCAGTGGTT
GGGCCTCTCTGGCAGGTGGGCGGTGTTGAGGCAAAGTCTTCGGTGTCCCCCGCCGGT
CTGGGCTTCGGTGTGGCGTGTAGGTTCGAGCTGAGCAGACGGAGGCTGTGCTTGACC
ATCGGTGATCAAAACTCCCTCTGCCCCCTGCCCAGACGCTCTAACATGCCCTCTGTCC
ATTTCCCTCTCCCCAAGGCCATGGGTTATAAAGGCCCTATGTAGGATGGGGAGCCAG
AGGCCCTAAGACATGAAGCACACCCCAGATCACTGTCTCTAGCCTTTCTGGGCACTG
AATCCATCCTGACCCACCACACACCCCCCGGCCAGTTGGCAAGAAAGAGGTGGCTCT
TGGGGGCTTTTATGCCCTTCATTAGCTGATGTTGGATTTTATATGCATTTTTATATTGT
CTCTAAGTGTCAGAACTATAATTTATTCATTTCTCTGTGTGTGTGTGTGCCAAGAAAC
GCAGGCTCTGGGCCTGCCTCCTTGCCCAGGAGGCCTTGCCAGCCTGTGTGCTTGTGA
GAACACATTGTACCTGAGCTGACAGGTACCAATAAAGACACTCTATTTTTAAAAAAA
AAAAAAAAAAAA-3'.
[0157] In addition, the L100 clone contains an ORF from basepair
145 through basepair 1890. This ORF encodes a polypeptide of 582
amino acid residues. The translational start site was assigned to
the first methionine residue in the ORF. The amino acid sequence of
the L100 polypeptide is as follows:
19 MAGIQKRKFDQLEEDDC (SEQ ID NO:27)
SSSSLSSGDLSPSPPSSSASPAWTSEEEGLGDQPPQPDQDSSGIQSLTPPSILKRAPRERPGH
VAFDGITVYYFPRCQGFTSVPSHGGCTLGMASRHSTCRLFSLAEFKQEQFRARREKLRRR
LKEEKLEMLKWKLSVSGVPEAGADVPLTVDAIDDASVEEDLAVAVAGGRLEEANFLQP
YPPRQRRALLRASGVRRIDREEKHELQALRQSREDCGCHCDGVCDPETCSCILAGIKCQ
MDHTSFPCGCCSEGCENPHGRVEFNQARVQTHFIHTLTRLQMEQGAESLGDPESPMEDV
PVEQTVVSPFPPSKPTMSNDLGDSSCGSDMTDSSTTYSSGGSGSRSEAPNHLAHPSL- PGSS
FRSGIDEDSLEQILNFSDSDLGIEEEEEEGGSVGNLDNLSCFHLADIFGTGDP- GSLASWTH
SQFGSSLPSGILDENANLDASCFLSSGLEGLREGSLPSSSGSPEGEAAQ- SSSLDLSLSSCDS
FELLQSLPDYSLGPHYTSRRVSGSLDSLETFHPSPSFSPPRDAS- FLDSLIGLSEPVTDVLAPL
LESQFEDTAVVPLDPVPV.
[0158] This amino acid sequence was found to contain numerous
cysteine residues, forming a motif that has features of a
methalothionein-like motif. Alignment analysis revealed that the
L100 methalothionein-like motif exhibits higher similarity with the
methalothionein motif from C. elegans than with the methalothionein
motif from mouse.
[0159] Northern blot and in situ analysis using a sequence from the
L100 clone revealed that L100 mRNA is weakly expressed in wild-type
rat brain. For in situ hybridization, Dig-labeled cRNA probes were
used as described elsewhere (Kuner et al., Science 283:5398
(1999)). Specifically, this weak L100 mRNA expression was observed
in the pyramidal cell layers as well as the dentate gyrus of the
hippocampus, thalamus, cortex, cerebellar granule cell layers, and
several fiber tracts including the fimbria hippocampus and the
cingulum. In addition, Northern blot analysis revealed that the
expression of the L100 mRNA was strongly upregulated in response to
the multiple MECS treatment. Specifically, L100 mRNA expression was
induced 17.2 fold by the multiple MECS treatment as determined from
Northern blot data using total RNA from rat hippocampus (Table
I).
[0160] The mRNA expression pattern of L100 demonstrated a
compelling overlap with neuronal populations known to release Zinc
into the synapse via synaptic vesicles and to take-up Zinc
post-synaptically. Briefly, synaptic release and uptake of Zinc may
participate in the induction and maintenance of epileptic seizures
and the neuronal cell death following epileptic seizures and
ischemia. The L100 metallothionine-like motif most likely enables
the L100 polypeptide to bind Zinc or other divalent cations in
vivo. The expression of L100 mRNA in Zinc-containing neuronal
populations in the brain indicates that L100 polypeptide may
sequester Zinc in brain.
[0161] In addition, when acute seizures were induced by kainate
treatment, the expression of L100 mRNA was strongly upregulated
(Tables II and III). Kainate-induced seizures is a model used to
study epilepsy. Briefly, 300-350 g male Sprague-Dawley rats were
intrapertoneally injected with either 10 mg/kg body weight of
kainate or PBS. RNA samples from the hippocampus, cortex, and
cerebellum were prepared from treated rats at 1.5, 6, and 24 hours
post-injection. This RNA then was used to measure mRNA expression
by Northern blot and RT-PCR analysis. Control mRNA measurements
included c-fos, GAPDH, NO-38, and ATF-4 for the Northern blot
analysis, and Hsp70, c-jun, Zif268, c-fos, Clathrin, and
.beta.-actin for the RT-PCR analysis. A Phosphoimager FLA2000
(Fuji) was used to analyze the data, which was expressed as the
Integral PSL-background PSL (ID evaluation with Aida version
2.0).
[0162] At six hours following kainate injection, strong
upregulation of the L100 mRNA was observed, by in situ
hybridization, in the dentate gyrus and areas CA3 and CA4 of the.
hippocampus as well as the associated entorrhinal cortex, the
cingulum, and fimbria, which are brain areas known to be highly
excited in and which mediate Kainate-induced seizures. Moderate
upregulation of the L100 mRNA also was found in the thalamic
nuclei, temporal, parietal, frontal, medial orbital, and cingulate
cortex as well as in the cerebellar granule cells. Thus, the data
presented herein indicates that L100 participates in cellular
mechanisms mediating kainate-induced epileptic seizures and the
consequent neurodegeneration.
20TABLE II mRNA expression normalized to GADPH expression 1.5 hour
1.5 hour 6 hour 6 hour 24 hour 24 hour Clone PBS kainate PBS
kainate PBS kainate Hippocampus: L100 4622 85251 7847 15444 3940
16551 L119 2816 69982 4597 11519 2787 12944 Cortex: L100 -- -- 81
290 86 131 L119 -- -- 255 1262 538 505
[0163]
21TABLE III Fold increase in mRNA expression upon kainate treatment
Hippocampus Cortex Clone 1.5 hour 6 hour 24 hour 1.5 hour 6 hour 24
hour A013 9.8 -- -- L094 3.6 -- -- L100 18.44 1.97 4.20 3.58 1.52
L119 24.85 2.51 4.64 -- R113 2.0 -- -- R286 -- -- --
[0164] In addition , when acute seizures were induced by
pentylenetetrazole (PTZ) treatment, the expression of L100 mRNA was
strongly upregulated (Tables IV and V). PTZ-induced seizures is a
model used to study epilepsy and ischemia. Briefly, 300-350 g male
Sprague-Dawley rats were intrapertoneally injected with either 50
mg/kg body weight of PTZ or PBS. Total RNA samples from the
hippocampus, cortex, and cerebellum were prepared from treated rats
at 20 minutes, 6 hours, and 24 hours post-injection. This RNA then
was used to measure mRNA expression by Northern blot analysis.
Control mRNA measurements included c-fos and GAPDH. A Phosphoimager
FLA2000 (Fuji) was used to analyze the data, which was expressed as
the Integral PSL-background PSL (ID evaluation with Aida version
2.0).
22TABLE IV mRNA expression normalized to GADPH expression 20 min 20
min 6 hour 6 hour 24 hour 24 hour Clone PBS PTZ PBS PTZ PBS PTZ
Hippocampus: L100 534 1637 854 1992 966 1903 L119 342 965 -- -- --
-- Cortex: L100 958 2719 1162 3740 1175 1825 L119 577 1605 -- -- --
--
[0165]
23TABLE V Fold increase in mRNA expression upon PTZ treatment
Hippocampus Cortex Clone 20 min 6 hour 24 hour 20 min 6 hour 24
hour L100 3.1 2.33 1.97 2.84 3.22 1.55 L119 2.82 -- -- 2.78 -- --
R113 -- 2.0 -- R286 -- 2.6 --
[0166] In another study, the expression pattern of L100 and L119
was determined using two models for ischemia. Briefly, neurons
degenerate in brain and spinal cord after acute insults (e.g.,
stroke, cardiac arrest, and trauma) and during progressive,
adult-onset diseases (e.g., amyotrophic lateral sclerosis, and
Alzheimer's disease). Impaired energy metabolism plays an important
role in neuronal cell death after brain ischemia, and apoptosis has
been implicated in cell death induced by metabolic impairment. The
irreversible inhibitor of succinate dehydrogenase in the
mitochondria, 3-nitroproplonic acid (3-NP), inhibits oxidative
phosphorylation and causes intracellular hypoxia. Thus, one model
used to study ischemia involves intrastriatal injections of 3-NP,
which is known to produce selective cell death similar to that
observed in transient ischemia and Huntington's disease (McLaughlin
et al., J. Neurochem 70:2406-2415 (1998)). The other model is a
global ischemic paradigm that involves a 15 minute insult by
complete occlusion of the carotis.
[0167] In the 3-NP study, 220-300 g Wistar rats were
intraperitoneally injected with 20 mg/kg body weight. Three hours
post-injections, the brain was removed and total RNA prepared. In
the global ischemia study, 220-300 g Wistar rats were received a 15
minute insult (bilateral occlusion of the Carotis/arterial
pressure=35 mm Hg). One hour later, the rats received a reperfusion
followed by immediate brain dissection and total RNA preparation.
Untreated rats were used as controls for each study. Ten (10) .mu.g
of total rat brain RNA (without cerebellum) was loaded per lane and
blotted. Probes were prepared from the 3' untranslated regions of
L100 and L119. The Northern blot data was collected using a
Phosphoimager (FLA2000 Fuji, Tina software) and expressed as
PSL-background.
[0168] L119 mRNA expression was upregulated 6-fold by global
ischemia while L100 mRNA expression was not inducible by global
ischemia (Table VI). This result indicates that only seizure
related stimuli alter the expression level of L100 and that L100 is
not a general marker for stress response of the cell like
c-fos.
24TABLE VI mRNA expression after 3-NP or global ischemia treatment.
Probe Untreated 3-NP Global Ischemia c-fos 18.1 26.64 216.22 GAPDH
487.02 587.51 593.31 L100 30.95 43.82 40.15 L119 55.48 41.94
332.73
[0169] Northern blot analysis using multiple tissues from rat
revealed that the expression of L100 and L119 mRNA was not brain
specific (Table VII). Briefly, fragments from the 3' untranslated
region of L100 and other IEG clones were labeled with
.sup.32P-dCTP. The denatured probe was hybridized with 10 .mu.g
total RNA from rat brain, liver, lung, muscle, intestine, eye,
heart, testis, and kidney in the Quik Hyb-solution (Stratagene) at
68.degree. C. and washed with 0.1.times.SSC at 60.degree. C. For
L100, after one day of exposure, signals were detected at the 3 kb
position in brain. In addition, a weaker signal was detected in
heart and a faint signal detected in kidney. A strong signal was
detected in testis but this signal was at a position corresponding
to a size smaller than 3 kb. For L119, a strong signal was detected
in heart and weaker signal in brain. In addition, only very faint
signals were detected in liver, kidney, and testis.
25TABLE VII mRNA expression in various rat tissues. Kid- Mus-
Intes- Probe Brain Liver Lung Heart ney cle tine Testis Eye A013
(+) (+) (+) (+) L094 + + (+) + (+) + L100 +++ ++ + +++(*) L119 ++
+++ R113 (+) (+) (+) (+) (+) (+) (+) R286 +++ (+) +++ (+) + (+) (+)
++ (*) smaller transcript
[0170] Another IEG nucleic acid clone was designated L111. The
first round of screening produced a clone (designated L111-5) that
contained a 3.0 kb fragment of L111. A second round of screening
using the coding region of L111-5 as a probe produced several
additional clones. The following nucleic acid sequence is within
the L111 clone:
26 5'-ATTCGGCACGAGCCAGAG (SEQ ID NO:28)
TGAAGGGGCATGGAGAAGTGGACGGCCTGGGAGCCGCAGGGCGCCGATGCGCTGCG
GCGCTTTCAAGGGTTGCTGCTGGACCGCCGCGGCCGGCTGCACTGCCAAGTGTTGCG
CCTGCGCGAAGTGGCCCGGAGGCTCGAGCGTCTACGGAGGCGCTCCTTGGCAGCCA
ACGTAGCTGGCAGCTCTCTGAGCGCTGCTGGCGCCCTAGCAGCCATCGTGGGGTTAT
CACTCAGCCCGGTCACCCTGGGAGCCTCGCTCGTGGCGTCCGCCGTGGGCTTAGGGG
TGGCCACCGCCGGAGGGGCAGTCACCATCACGTCCGACCTCTCTCTGATCTTCTGCA
ATTCCCGGGAGGTACGGAGGGTGCAAGAGATCGCCGCCACCTGCCAGGACCAGATG
CGCGAACTCCTGAGCTGCCTTGAGTTCTTCTGTCAGTGGCAGGGGCGCGGGGACCGC
CAGCTGCTGCAGAGCGGGAGGGACGCCTCCATGGCTCTTTACAACTCTGTCTACTTC
ATCGTCTTCTTCGGCTCGCGTGGCTTCCTCATCCCCAGGCGTGCGGAGGGGGCCACC
AAAGTCAGCCAGGCCGTGCTGAAGGCCAAGATTCAGAAACTGTCTGAGAGCCTGGA
GTCCTGCACTGGTGCCCTGGATGAACTTAGTGAGCAGCTGGAATCCCGGGTCCAGCT
CTGTACCAAGGCCGGCCGTGGTCACAACCTCAGGAACTCCCCTGATCTGGATGCAGC
GTTGTTTTTCTAAGAGCATCCTCTAGCTGTGTGGAATGTTCTAGATTCGCAGCATCCA
CAAGGAAGTGCTACATGGGCGGAGTGCAAAGGATTTCAGAAGCTCTTCTTGCAGGG
CATCAGTCCGTAGCTCCTTGTGTGTGCGAAAGACTTTTCACTTGTGTAATCCCAACTG
AGTATGTGACCCTAAACAGTCACTTTGGGGACTCCCCAAATCCTTTTTAGCTGCACA
CAGCTTGTCAGACTGTCCTTCAATTAGAGTTATTGGGGTGGGGGGGCTTGATGGCTT
GAGTAATAGAGGTCTGGCGAGGTGTCTCCCTCTTGGACCTCTTATGTGTTGTTACTAG
AATCCTGAGATTCTCAAATGTTGGTGAGAGGAGACTTTTACTTTTCAACTTTGCTTCG
GCAGTTTCCGATACACAGGACTCCAGAATCCAGAACAAGAAAGAAGAACCTTGTGT
TTGTAGGGTGTGCAGACCCAGACGGGGCCGAGGAGCTGACTTGCTCAGCTCTCACAC
GCAGCCAGTTTATCCACTCACAGACCAAACCTGGCTACTGCATAGACTGTTCCAGTG
TGGCTTCAAATCCACACCTCTAGGTACCCTGAGAAGGAAAGCCACCTGAAGAGTCA
CTCTAATCCCAACACGCTCACCCCCTTCACGTCCATAAAGGAGCTGGGCAAGGGGTG
AGATGAAGACCCTGACAATTTTAAATGACTGTAGCATAGAGAGCCATGGCCTTTGAG
TTTAAGAGTCTTGATCCCAGGTTCTGTCCCCCACTGTCCTGTGACTTAGCCACCTTGT
CTTGCTACAGATGGTGGTAGGAGGCCACCCTGTTGCGAAGTCCTGAGATAATGACAA
ACACAGAGGCTAGCTCACAAAAATGTACTTCCTGGCCTGGCTTCTGAAGGGTTAACT
GTTGGGCTCCATCCCAGATTTCTGAGATCAGGAACTCCAAATATGAGGCCCGCCTCT
GGCTGATTCTGATGCCCCATAAATGTTTGAAAATGACACAGCAAAGGTTCATCTCCA
GCCAGGTGTGGTGGGACACACCTGTAAGGCCAGCGCTTGGAGATGGAGACAGGGGG
ACCAGTAGTTCAGGGTCATTCTTGGCTACATAGCAAACTCAAGGCCACCCTGGTCTC
AAAAACCAAAACAAAAAGCCATCTTCTGACTCCCTTCAATTGTTCAAAGCCTTTCCA
GGGCCTTCAGAATCACGCTCAGAGTGTTCTGGGAAGATTAGCCCAGAAGCCAGAGA
AAGAGTACGCTGTGTGCTTGTAAAGCCAGTTACTCTGTCCCCTGTGAACTAGGAGAC
AGAGCACTTCCGACCCTATAGAGGGCAGTAGTGGCCATTCCTTGTAGGGGACTGGTA
TAGAAGTAATGTGAACTATTTAAAAATAGTTATTTAATTGCTGCCTTCACATTTGATT
TTATTTAACCTTCACATTATTTAGAAAATAATAAGAGTAGTAAGTGTCTGAATAGGA
AGGGAGTCTCTTAAGGCTCTTTCCAAGAGCTCAGGTTTGGATTTCTAGAGTCCCCCC
GACCCCAGAGAGGACTCTTTAGTGTTTGACACGGTCTTTGTAAGTAAGATGGGGAGT
CCTGGAGAGAGAGACCAAGCTGATTTTTAAACTAGGAAATGGAGTCTTGAACTGTG
GAAGATTTGAAAAGTTAAGCCTATGTGTCTTGAAGGTACTTGGCCAGAAAAGCACTT
GGCTTGAAAAAGAAAACCTGTTTAATTCAGGGGTGGAGGAATAGAGACAGATGAAG
AAAGCATTTAGACCTCGGAAACCTGATGTCCTATGAAATTCTGTTTTTATAAAATTGT
GTTATGGTGGAGATCTGTTGCATTTCGACTTTGTGGCTGTAAGAAACCTGTTATCTAT
GTTTAAGAAAGTACTTCTAATTTATTCAATGTCTTCCTAAATTATCCTTTAAAAAAAA
AAGTTGGAAAGTCTATGAGACCGTACCTAAGAAACCTTGACTGTGTATTTAAGTTAT
TTAATGCCATGCATTTGTGAAGCCCCTTCCCAGTGATGGCTGTGGTGTGTCTGAGGA
AATGTAAGTTTGGCATGAGGGGGAGGGGCTGCTGTTTCTATATTTGTTTTTGTTTTCT
ATAAACAGTAATCAGGATGTATCCTGGTTTCATTTGACATTGAAAAAAAAAAAAAA
ACTCGTGCCGAATTC-3'.
[0171] The L111-5 clone contained 0.5 kb of the 3'-end of an
ORF.
[0172] Northern blot analysis using a sequence from L111 revealed
the presence of a 4.0 kb mRNA transcript. This analysis also
revealed that the expression of L111 mRNA was marginally
upregulated in response to the multiple MECS treatment.
[0173] Another nucleic acid clone was designated L117. The L117
clone is 2460 bp in length and has a nucleic acid sequence as
follows:
27 5'-TACGGCTGCGAGAAGACGACAG (SEQ ID NO:29)
AAGGGGAGCGGAGCCAAGATGGCGGCGGAGCTGGAATACGAGTCTGTGCTGTGTGT
GAAGCCCGACGTCAGCGTCTACCGGATTCCGCCGCGGGCCTCCAACCGCGGTTACAG
GGCATCTGACTGGAAGCTAGACCAGCCTGATTGGACTGGTCGCCTCCGAATCACTTC
AAAAGGGAAGATTGCCTACATCAAACTGGAAGATAAAGTTTCAGGGGAGCTCTTCG
CTCAGGCGCCAGTAGAGCAGTACCCTGGGATTGCTGTGGAGACTGTGGCCGACTCCA
GCCGCTACTTTGTGATCAGGATCCAGGATGGCACCGGGCGCAGTGCGTTTATTGGCA
TCGGCTTCACGGACCGGGGAGATGCCTTCGACTTTAATGTCTCCCTGCAAGATCACT
TCAAGTGGGTAAAGCAGGAAACCGAGATCTCCAAAGAATCGCAGGAAATGGATAGT
CGTCCCAAGTTGGATTTAGGCTTCAAGGAAGGGCAAACCATCAAGCTGAGTATTGG
GAACATTACAGCCAAGAAAGGGGGTACTTCTAAGCCCCGGGCCTCAGGAACGGGGG
GCCTGAGCTTACTCCCACCTCCTCCTGGAGGCAAAGTCACTATCCCCCCACCGTCCTC
CTCCGTTGCCATCAGCAACCACGTCACCCCACCACCCATTCCAAAATCTAACCATGG
AAGTAATGATTCAGATATCCTGTTAGATTTGGATTCTCCAGCTCCTGTCCCGACTTCA
GCACCAGCTCCAGCTCCAGCTTCTACAAGCAATGACTTGTGGGGAGACTTTAGCACT
GCATCCAGCTCTGTTCCAAACCAGGCACCACAGCCATCTAACTGGGTCCAGTTTTGA
GTCGCATTGGCAAGAAGTTGAGGACACTTGAAGAATAAAAATGACCTCAAGGGCAC
CATTCTATGAGGGAGTTGAGGGACGGCTTAATTTCCCAGGACCCAAATCAGTGGTCA
GTCTTTCCTGTAGCTTCTCTGTGCATTCAGGCTGGATTTTTTTTTTTTTTTTTTTTGGTT
ACCTCTGTGTTACTTGCTGTATATCCAGGAGACAATCTGCTGTTTCCTGCTCAGAACC
AAGCAAGGGAGTAGTGGGTATTATCACACTGACTGACTTTGCAGAGTTCAGAAGGC
CAACTTGATGAGTGGGAGTGACCTCGAACGTATGTAAATCCTTGAACTTATTTCAGA
ATCATCTCATGATTCCCTAGTTAGCAATTTCAGGAGAGACAAATGCCTTGAAACTGT
CTTCTCCACTAATCCGAGACTAAATATGGTCAGGCTGGCCCCAGGACTCATGAAGTT
AGGGTTTTCATGGGGGTAGATTTGGAGAAAGCTGTGTCCCGGCTCTCTTCTGTAAGG
CCTCCTTCAGGCTTACCCCATGCAGTGAACTTCCCGTGCTGGGTGGAGCCCCATCAC
CTTCTTGTGTGTTTACATGTTGTTTCCTTTGACAAGAGGGTTATGTTGGTGGCACCTC
ACTGTTTTCTTGTTGAATAGTGCAGCATCTTTGACCAGTGAATATTTCTGAGATGAAG
GGGTCAAGGGGCTGTGCTTTCCATGGTGTAGTCTACAGAAGTGTTTAATTTCTTGCG
GCCCCACGGGATTGCTGCACTGACGCATAGAATTGATCTATACTCACCCTGTGTTTG
ACCTGAAGAGTTTTAACTTGATGTGTAGAGCAGAGAGCTGGAAGCACTAAGTTCCCA
TTCAGTACCCACAATGCCTTGCTGCCTGGTTTGACTCCTTTTCATAAACATTTCATTT
CAGTCCATCTAGCACTTCTGTGGAAAGCTGCTGTTGATTGTGTCAGTGTGAAGGAGG
TGAAGTCACAGCTTTCTTTACCTATGACAGTTAGGCTTTGCACTAGACGTTGATACCA
GCTAGGATATCTTAAAGGAAGTTACCGCCCCATCACTCTCCAGTCTCTGGCCGCCAT
TCCTTTTACAGTGCTGTGAAGAGCGTCCTCTGAGGTCGGTGGGTACTGTCTCCTGTTG
GTCGGGCAGTTTGAGGGAGGAGTGGGAGGACTCACACTCCTGCAGGTACCTGTTTG
GGTAGCACACTGGCTGCAGAGAGTCCTTTCAGATATATTGTTTCTCAATGTTCTTCGT
AGCTTTTTCTAACTTCGGGTCCATTTTTCCCATCGCCTCTTCCCATTCCCAGGCAGCTC
TCTTGTTGCAGAGCCATGGCAGGACGTTTAAGTTCCAATAAAAACACTAAGAAGAA
AGTATAGAATCACTAGTGACTGTTGGGAAACCTATTTTCTCAATCTTCCTCCATTTTG
TGTTCTTTGTATTCTTAAGATGATAATATATTATGTATTTGAATTGCTGAAAATTGAA
AATGAAGTTGAAGATATATGTATATAAGCGTATGCTGTATTGGTGCAATAATGGTAA
TTAAAGATATTAAAAAAGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA-3'.
[0174] In addition, the L117 clone contains an open reading frame
(ORF) from basepair 42 through basepair 875. This ORF encodes a
polypeptide of 278 amino acid residues. The amino acid sequence of
the L117 polypeptide is as follows:
28 MAAELEYESVLCVKPDV (SEQ ID NO:30)
SVYRIPPRASNRGYRASDWKLDQPDWTGRLRITSKGKIAYIKLEDKVSGELFAQAPVEQ
YPGIAVETVADSSRYFVIRIQDGTGRSAFIGIGFTDRGDAFDFNVSLQDHFKWVKQETEIS
KESQEMDSRPKLDLGFKEGQTIKLSIGNITAKKGGTSKPRASGTGGLSLLPPPPGGKVTIP
PPSSSVAISNHVTPPPIPKSNHGSNDSDILLDLDSPAPVPTSAPAPAPASTSNDLWGDFSTA
SSSVPNQAPQPSNWVQF.
[0175] Using tblast2x algorithms, the L117 polypeptide was found to
have homology with expressed sequence tags (ESTs) from mouse, mouse
embryo, human hNT neurons, human tumors, drosophilia, drosophilia
embryo, C. elegans, and Arabidopsis thaliana, a plant organism.
Although the sequence of ESTs can be questionable, the identified
ESTs were aligned for comparison. The comparison of consensus
sequences from each species provided evidence that the L117 clone
or a L117 motif has a very strong pressure for conservation during
evolution since it is conserved in a variety of very distant
species. In addition, this alignment indicated that the first
methionine residue in the ORF of the L117 clone is the true
initiation site for translation since most of the homology between
the ESTs begins around this position, and the C. elegans,
drosophilia, and human hNT ESTs each contain a methionine residue
that is in a very close proximity to that of the L117 clone.
Further, the relation between these ESTs and the L117 clone was
supported by an exactly matching stop codon in the human EST, mouse
EST, and L117.
[0176] Northern blot analysis revealed that the expression of the
L117 mRNA was not upregulated in response to the multiple MECS
treatment in either the hippocampus or cortex. Analysis using a
total RNA extract, however, revealed a small upregulation upon MECS
stimulus.
[0177] Another IEG nucleic acid clone was designated L119. The L119
clone is 2900 bp in length and has a nucleic acid sequence as
follows:
29 5'-ATTCGGCACGAGCCAGAG (SEQ ID NO:31)
TGAAGGGGCATGGAGAAGTGGACGGCCTGGGAGCCGCAGGGCGCCGATGCGCTGCG
GCGCTTTCAAGGGTTGCTGCTGGACCGCCGCGGCCGGCTGCACTGCCAAGTGTTGCG
CCTGCGCGAAGTGGCCCGGAGGCTCGAGCGTCTACGGAGGCGCTCCTTGGCAGCCA
ACGTAGCTGGCAGCTCTCTGAGCGCTGCTGGCGCCCTAGCAGCCATCGTGGGGTTAT
CACTCAGCCCGGTCACCCTGGGAGCCTCGCTCGTGGCGTCCGCCGTGGGCTTAGGGG
TGGCCACCGCCGGAGGGGCAGTCACCATCACGTCCGACCTCTCTCTGATCTTCTGCA
ATTCCCGGGAGGTACGGAGGGTGCAAGAGATCGCCGCCACCTGCCAGGACCAGATG
CGCGAACTCCTGAGCTGCCTTGAGTTCTTCTGTCAGTGGCAGGGGCGCGGGGACCGC
CAGCTGCTGCAGAGCGGGAGGGACGCCTCCATGGCTCTTTACAACTCTGTCTACTTC
ATCGTCTTCTTCGGCTCGCGTGGCTTCCTCATCCCCAGGCGTGCGGAGGGGGCCACC
AAAGTCAGCCAGGCCGTGCTGAAGGCCAAGATTCAGAAACTGTCTGAGAGCCTGGA
GTCCTGCACTGGTGCCCTGGATGAACTTAGTGAGCAGCTGGAATCCCGGGTCCAGCT
CTGTACCAAGGCCGGCCGTGGTCACAACCTCAGGAACTCCCCTGATCTGGATGCAGC
GTTGTTTTTCTAAGAGCATCCTCTAGCTGTGTGGAATGTTCTAGATTCGCAGCATCCA
CAAGGAAGTGCTACATGGGCGGAGTGCAAAGGATTTCAGAAGCTCTTCTTGCAGGG
CATCAGTCCGTAGCTCCTTGTGTGTGCGAAAGACTTTTCACTTGTGTAATCCCAACTG
AGTATGTGACCCTAAACAGTCACTTTGGGGACTCCCCAAATCCTTTTTAGCTGCACA
CAGCTTGTCAGACTGTCCTTCAATTAGAGTTATTGGGGTGGGGGGGCTTGATGGCTT
GAGTAATAGAGGTCTGGCGAGGTGTCTCCCTCTTGGACCTCTTATGTGTTGTTACTAG
AATCCTGAGATTCTCAAATGTTGGTGAGAGGAGACTTTACTTTTCAACTTTGCTTCG
GCAGTTTCCGATACACAGGACTCCAGAATCCAGAACAAGAAAGAAGAACCTTGTGT
TTGTAGGGTGTGCAGACCCAGACGGGGCCGAGGAGCTGACTTGCTCAGCTCTCACAC
GCAGCCAGTTTATCCACTCACAGACCAAACCTGGCTACTGCATAGACTGTTCCAGTG
TGGCTTCAAATCCACACCTCTAGGTACCCTGAGAAGGAAAGCCACCTGAAGAGTCA
CTCTAATCCCAACACGCTCACCCCCTTCACGTCCATAAAGGAGCTGGGCAAGGGGTG
AGATGAAGACCCTGACAATTTTAAATGACTGTAGCATAGAGAGCCATGGCCTTTGAG
TTTAAGAGTCTTGATCCCAGGTTCTGTCCCCCACTGTCCTGTGACTTAGCCACCTTGT
CTTGCTACAGATGGTGGTAGGAGGCCACCCTGTTGCGAAGTCCTGAGATAATGACAA
ACACAGAGGCTAGCTCACAAAAATGTACTTCCTGGCCTGGCTTCTGAAGGGTTAACT
GTTGGGCTCCATCCCAGATTTCTGAGATCAGGAACTCCAAATATGAGGCCCGCCTCT
GGCTGATTCTGATGCCCCATAAATGTTTGAAAATGACACAGCAAAGGTTCATCTCCA
GCCAGGTGTGGTGGGACACACCTGTAAGGCCAGCGCTTGGAGATGGAGACAGGGGG
ACCAGTAGTTCAGGGTCATTCTTGGCTACATAGCAAACTCAAGGCCACCCTGGTCTC
AAAAACCAAAACAAAAAGCCATCTTCTGACTCCCTTCAATTGTTCAAAGCCTTTCCA
GGGCCTTCAGAATCACGCTCAGAGTGTTCTGGGAAGATTAGCCCAGAAGCCAGAGA
AAGAGTACGCTGTGTGCTTGTAAAGCCAGTTACTCTGTCCCCTGTGAACTAGGAGAC
AGAGCACTTCCGACCCTATAGAGGGCAGTAGTGGCCATTCCTTGTAGGGGACTGGTA
TAGAAGTAATGTGAACTATTTAAAAATAGTTATTTAATTGCTGCCTTCACATTTGATT
TTATTTAACCTTCACATTATTTAGAAAATAATAAGAGTAGTAAGTGTCTGAATAGGA
AGGGAGTCTCTTAAGGCTCTTTCCAAGAGCTCAGGTTTGGATTTCTAGAGTCCCCCC
GACCCCAGAGAGGACTCTTTAGTGTTTGACACGGTCTTTGTAAGTAAGATGGGGAGT
CCTGGAGAGAGAGACCAAGCTGATTTTTAAACTAGGAAATGGAGTCTTGAACTGTG
GAAGATTTGAAAAGTTAAGCCTATGTGTCTTGAAGGTACTTGGCCAGAAAAGCACTT
GGCTTGAAAAAGAAAACCTGTTTAATTCAGGGGTGGAGGAATAGAGACAGATGAAG
AAAGCATTTAGACCTCGGAAACCTGATGTCCTATGAAATTCTGTTTTTATAAAATTGT
GTTATGGTGGAGATCTGTTGCATTTCGACTTTGTGGCTGTAAGAAACCTGTTATCTAT
GTTTAAGAAAGTACTTCTAATTTATTCAATGTCTTCCTAAATTATCCTTTAAAAAAAA
AAGTTGGAAAGTCTATGAGACCGTACCTAAGAAACCTTGACTGTGTATTTAAGTTAT
TTAATGCCATGCATTTGTGAAGCCCCTTCCCAGTGATGGCTGTGGTGTGTCTGAGGA
AATGTAAGTTTGGCATGAGGGGGAGGGGCTGCTGTTTCTATATTTGTTTTTGTTTTCT
ATAAACAGTAATCAGGATGTATCCTGGTTTCATTTGACATTGAAAAAAAAAAAAAA A-3'
[0178] In addition, the L119 clone contains an ORF from basepair 28
through basepair 768. This ORF encodes a polypeptide of 247 amino
acid residues. The translational start site was assigned to the
first methionine residue in the ORF. The amino acid sequence of the
L119 polypeptide is as follows:
30 MEKWTAWEPQGADALRRFQGLLLDRRGRLH (SEQ ID NO:32)
CQVLRLREVARRLERLRRRSLAANVAGSSLSAAGALAAIVGLSLSPVTLGASLVASAVG
LGVATAGGAVTITSDLSLIFCNSREVRRVQEIAATCQDQMRELLSCLEFFCQWQGRGDR
QLLQSGRDASMALYNSVYFIVFFGSRGFLIPRRAEGATKVSQAVLKAKIQKLSESLESCT
GALDELSEQLESRVQLCTKAGRGHNLRNSPDL DAALFF.
[0179] Hydropathy plot analysis revealed a stretch of about 50
hydrophobic amino acid residues, possibly indicating that the L119
polypeptide is a type II transmembrane protein.
[0180] Northern blot analysis using a sequence from the L119 clone
revealed that the expression of the L119 mRNA was strongly
upregulated in response to the multiple MECS treatment.
Specifically, L119 mRNA expression was induced 17.8 fold by the
multiple MECS treatment as determined from Northern blot data using
total RNA from rat hippocampus (Table I).
[0181] Another IEG nucleic acid clone was designated R010. The R010
clone is 1280 bp in length and has the following nucleic-acid
sequence:
31 5'-GCTTTGGAAACCGGACTGCAGGCT (SEQ ID NO:33)
AAACTGGCTTCTTTTGAATCCTTGGAAGCATAAAGGACAAGTAGCAGGGCTCGCAGT
CTTCCATTTGTCACTGGAGAAGAACTTATAATTCAGAAGATCTGGGTCTGGACCCAG
GCTGACCACTTTGGAGCTTTGAGACTCTGGGATTGTGATCCAGTTCTGAGCTGGTGA
TAAACACTCCTTGTGACTTTTGGTCAATTCAGCTACCAGATTCCAGCCAACATGACC
CTCGCAGCCTATAAGGAGAAGATGAAGGAACTCCCACTAGTGTCTCTGTTCTGCTCC
TGTTTTCTGTCTGATCCCCTGAATAAATCATCCTACAAATATGAAGGCTGGTGTGGG
AGACAGTGTAGGAGGAAAGGTCAAAGCCAGCGGAAAGGCAGTGCTGACTGGAGAG
AAAGAAGAGAACAGGCAGATACGGTAGACCTGAACTGGTGTGTCATCTCTGATATG
GAAGTCATCGAGCTGAATAAGTGTACCTCGGGCCAGTCCTTTGAAGTCATCCTGAAG
CCACCTTCCTTTGACGGGGTGCCTGAGTTTAATGCCTCCCTCCCAAGACGTCGAGAC
CCATCGCTAGAAGAGATACAGAAGAAGCTAGAAGCAGCAGAGGAGCGAAGGAAGT
ACCAGGAAGCTGAGCTCCTAAAACACCTTGCAGAGAAACGAGAGCATGAGCGTGAG
GTAATCCAGAAAGCTATCGAGGAAAACAACAACTTCATCAAGATGGCGAAAGAGAA
GCTGGCCCAGAAGATGGAGTCCAATAAGGAAAACCGGGAGGCCCATCTGGCTGCCA
TGTTGGAGCGGCTGCAAGAGAAGGACAAGCACGCAGAGGAGGTGCGGAAAAACAA
GGAGCTGAAGGAAGAGGCCTCCAGGTAAAGCCCANAGGCCAAGGAAGTTTCCAGGA
CAGCCGGACAGCTCCCGCAGCAACCTGGTTCCAGCAGCATCGGCCGCTGGCTGCTCT
CCCAGCACTGGGGTTCGGGGGGAGGGGGGTGGCCAAAGGGGCGTTTCCTCTGCTTTT
GGTGTTTGTACATGTAAAAGATTGACCAGTGAAGCCATCCTATTTGTTTCTGGGGAA
CAATGATGGGGTGGGAGAGGGGACAGAGAGTGTTTGGAAAAGGAGGTGAAGATGA
GCCCGAGGACTTTGTGACACTGTCCACTGACTGCAGACTTGGGCCAAGGCCCCCGCT
TTTCACGGCTCTGCCTGGACATTCGGCCTCCAGGTTCCTAGTGGAGAGAAGATGTGA
CAGAAGTTCAGAGTGAAGGGCCGAGTCCTGGTGGGGTGGTGTGCAGGGCCAGCAGG
ACGAGCCCGTCTGGATGGAGTGAAACCTACCCTGAGCGGGTGGGATAAGGTCTGTG
TGCGTCTGTTCATTGTCATCTTTTGATCATCATGACCAACGAAACATTTAAAAAAAA
AAAAAAAAAAAAAA-3'.
[0182] Two genomic R010 clones were also obtained. The nucleic acid
sequence for these genomic R010 clones is as follows:
32 5'-GATAA (SEQ ID NO:34) ACACTCCTTGTGACTTTTGGTCA-
ATTCAGCTACCAGATTCCAGCCAACATGACCCTCG
CAGGTAGGTACATGCACCAGTCAGTGATGAACACCATAACACAAGCCATTTTTCTAT
CTCTGTGTGTGTCCATGTGTATTAAGGTGCATCCGTGTGTGTGATACACACGTAGGT
GCATGGCATGCATGTGTGTGCAAATGCATATACAAGTCCAAGGACAGGGGTTGGGG
ATTTAGCTCANTGGTAGAGCACTTGCCTANGAAGCGCAAGGCCCTGGGTTCGGTCCC
CAGCTCCGAAAAAAAGAACCAAAAAAAAAAAAAAAAAAAAAAAAAAAATTTCCAN
GGACAACCCCAAATTTCCTTTCNCNAAANCCANCCANCTTCCATTNAAAAAAAANG
GGTCNCNCNTGGGTTAAACCATTTNNAAANGGCNAACCTNACNGGCCAKTGAKTGC
CAGGAATCTTCTTATYCCTGCCCWACCTCCAATGTCTTTCACATGTGAATGCTGAGG
GTCAGAACTTGTGCTTACAAGGCAGACATTTTGCCAGCTCTCCGGCCATCTTTCTCTA
TGTATGTACACTCACAGATGCACAGGAAGAGAGGGTAGAGAAGCCAAGAGGCAAA
GTCATTTCTGGGTGGTGGGTGGGATCACAGCTGAATTCTTCTTCCTCATTTGCTCTGT
GTGTATTATTTAATTTTAAAATAATACCTTTATAATAGTATCGAAACTATGCTTTCAA
GTTTGTAAGAGAAAGTGATCACTGGGCTGTGTAGTGAGGGGGTCTTTATATTATGCA
TATAACATGGTGCAATGGGAAGGACTGGCAGAGGCCTCCATGATGACCTATGACTTC
TAGGGAGACTCAGTCGTGTCAAGGGTACATTCCTACTCTGCAGACAGCTTCTCCCTG
GTTTGATTCCTGTGCTGGGAAGATTTGAGGAGTCTTCCAGCCTGACCTCTTCTACAGT
GGGCCTGGACTTTAAGGAGAGTAGCAAGGAAGTCTTTTTATTAATCTCTTACCCTTT
AGGCAGCAGTGTCAAGTACTTTTAGCAGAATTAAATATAGATTTCCTACAAACTACA
AACTTCAAAGCCCTGGTTTATCCTTGGGTGGGAGTAGGAGATGGAGGGCCAGGGTC
AGGGCACTGCACTTGGGATCTTTACTTGAGGGTACTCAACGCTTGGTAGTAACAAAA
AGTGGGGTGAGTGACAATGTTAATTTTCAACTGGGAGGTAGCCCAGGCTTGGGTACT
TTGGAGCCAGAAAGCCTGGGCTGACTCACAGAAGTGGTGCTCTCTCTYGYAGCCTAT
AAGGAGWWGATGAAGGAACTCCCACTAGTGTCTCTGTTCTGCTCCTGTTTTCTGTCT
GATCCCCYGRATAAATCATCCTACAAATATGAAGGTGAGTAGGGGCTAGGCTGGGA
TAGAAAAGGGTGGAGGCTTCTGTGTCCTGTGTTTGTSGGTGCCCCACATTGACTCCTA
TCTTGTAAAACTGTCCTGGTCGCAGTGTGTCTTATTTCCCAGAGGCTGAGGAGTCTG
AGCCCAGGGGGATGTAGCCTGGGTGCCAAGCAGCCTCCAGGGATCTGGATTGGGCC
CTCCTGGAGCACTTGCTCCTAGAGTCCCTTTTRCACATTCCTTGACACCACAGAGGAC
ACCAGGATAAGCCAGACACAAGTTTTGAGATTCCATTCATGGAGGCCCAGAACAGA
AAAAGAAAACTTAGTGTGTTCACCAGGGCTTCTAGGGACAGGTAGAGATGCTCCTA
GACAGGTCCAGGGTGGGAATAGCACTTCTAACCTGGATGGTGACAGTTCGAGCCCCT
AGACCCTATCAGAGAGTACTGGATTGTCATGCTGTCAGGAGGAGTGGTCAGGGGAC
AGATAGGTCATCTCTTCATTTCTGTTTGCCAGGAAGGGATGGGTTTGGTCTGTCAATA
AGAGAGATGGGTGTTTGGATGACCTGAGTCTGTTTTTTCCATTTAGGCTGGTGTGGG
AGACAGTGTAGGAGGAAAGGTCAAAGCCAGCGGAAAGGCAGTGCTGACTGGAGAG
AAAGAAGAGAACAGGGTAGGCCGGAGCCAGGGGAGAGGTCCACAAGCCATCAGAG
GGACAGGGCAAGGAGGGGCTGGCGGTGGGGATGGGTGAAATGAACTGGTGTCTGTC
ACCAGCGAGGAACAACAGCAGCTGGTGCTATCACAAATCACAGCTCCCTGCTTACCC
TGTAAAAGCCATTGACCTTAGGGTCCAACGTTCAGGATCGACCAGACCCCTAGTCAT
TGGTGTGCCTTGGGACCCTCAGCTTTCCTGTGTCTGTGTGCATGTACACATGCTCATT
GGGGCCCCAGCTGCTCCTCAGAAGGTGAGCAGCCCCAACTCTGCCCTCCATAGCAGA
TACGGTAGACCTGAACTGGTGTGTCATCTCTGATATGGAAGTCATCGAGCTGAGTAA
GTGTACCTCGGGCCAGTCCTTTGAAGTCATCCTGAAGCCACCTTCCTTTGACGGGGT
GCCTGAGTTTAAAGCCTCCCTCCCAAGACGTCGAGACCCATCGCTAGAAGAGATACA
GAAGAAGCTAGAAGCAGCAGAGGAGCGAAGGAAGGTTAGTGTAGCCCCATGTCACT
TCCTCCCATCCCAGCGGGAGCAGGAAGTGCAGCTCCATATCTCTTCCTCCCATCCCA
GTGGGAGTGGGAAGGATATTTAGACAGCACCTCCTGAGTGCTGGGCATAGACCGGT
AGTTCTCAACCTTCTTAGTGCTGTAACCCTTAATATATATATATATATATATATATAT
ATATATATATATAGTTCCTCATGTTGTGATTACCCCCCATACCATAAACTTATCCCGT
TGCTCTTTATGTCTTCATAATTATAATTTTGCTACTGTTATGAATTGTGATACAACTAT
CAGACCTGCACCCCCTAATGGCAGCAGCCCACGTGTTGAGAACCACTGGCATAGAT
GTAGACTAAGATACCACCTGAAGGGGACAAGACTATGACTATGCACTGGGTGAGCT
TACAGTGTGGCTAATGGCTAAAATGTCACAGTCCTCACAAAGCTGCCTTTGTATGCA
GCTTCCTTGTTCCCCATTGATTCTMGTCCSTCAGCTCAGATGCCCATTTTAATGTGAG
TGTTTCTTNACCTTTCAGAAANACAAAACAAAACAACCCAGCTTTCTCCACTNAATT
GTGTGGTCCCTCCCTTTAAATATCCAAAGCATTTATCACACCCAGGTCTGGNGTCCA
NTATNTATTGATATGCGTGTTTATTTNNACTAGGGCAATTNTCTCCNTTCCCTGGTGT
CTGGAGTTGTGAGGGCCTTGAGGTTTATAGAAGATCACTTAGTACTTGTGAATGAAC
GCGAGGAAAAGGAGAAAAGAGACTCAGAAGCTACTTNGGAAAGGGCTACNAAAGC
CAAATATGACGGAAAGGTTTGCAGTCCATGNCGTTGTTCTCTGCTTCTGGGACAGAG
GACCAGGTTCATCTCATCTGGGCATGGCACTGTTCAGCTGTGGTGGTAGAAATCCAC
TCTAAAGGGTCNTTCTCTTTCTTTTGNTGCCCTAGTACCAGGAAGCTGAGCTCCTAAA
ACACCTTGCAGAGAAACGAGAGCATGAGCGTGAGGTAATCCAGAAAGCTATCGAGG
AAAACAACAACTTCATCAAGATGGCGAAAGAGAAGCTGGCCCAGAAGATGGAGTCC
AATAAGGAAAACCGGGAGGCCCATCTGGCTGCCATGTTGGAGCGGCTGCAAGAGAA
GGTAAGAGGTCCTGGATTGGCAGGAGGCTCCTTCCATGGCAAGAACGTGCAACCTA
CACATCACTCTGGAGGAAGCGGCCTATGCAGGAATTGAAATGTTTCTACCAGGCAG
GGTCCTCATTGTTCTAAGGGGAAGATTTGGGAAGTCATAGGCAAGAAGCTCACACC
AAACCCTGGGTGGCCTCCGGGGATCTTCTANGGTTTTGAACCGGAAATTCTGCACTG
TCTCANGAGCTTGCTCACACCCTTCTTTTCTAAAGAAAGCCCGCCCAGTGCAAGTAT
CTAAGGAGAGGCACATGTCTACACATTTCTGGCTTCATCATTGAATGGGCAGATTTG
GGTTAGTGAAAGATACAGTCAGCTTGGCTTTGAGCCANGGATACAGCAAGCTCGGTT
GCCAATACAGCAGGATACAGGATTCTCCCCAGAGCTCCTCGTAAGGGCCAGAGAGT
ANTAGGTTTTCCTCAATAGTCTGCCTTTGTCAATAACTCAAATGTCACCTGCATCTGA
GCGGTGTGCGAGACTGGGGTTGGTCCTCCATGTTATTCTTTGGAAGACGTGCTGACC
TCATTTCCTGAGTCCCAGGCTGCCTACGTTTCTCCTGCAGCTCCTGGGAAGCTTTAGC
TCTGTGTTTTATTTCCAAGGAGCCGCCTGCTGCGCGGTGACTCCCGGGACSGATCGGT
GGCCTCGTCCCATGGTGAGCAGCGTGGTCCTTATTCCTTCCTGCCTACCCACCTAAAA
CCTCAGGCCCTTGACAATTACCACAGAAAGATCTGGCTTCATCCAGGGATGTGAGCA
GCACAGGCTGGCCAGTAGGTGGCAGCCCTGTGCTCATGTTCAATTACAGGAGGGAC
AGCAAGGCTTCTTTCTCCACTGAGTGCCTTGGGGGAGGGACACAATCTGAGTGTGAC
TTTGGGCTCCTCCAGTTAATGAGAGATACTGTAAGAAAACTTAAGATTGCCTTTACT
TTTTATACCAGGTCTCATGCATTCCAGGCTGGCCTCAAATTGGCTAAATTGCTGAGG
CTAGCCTTGGAATTCTTATCATTTTGTCTTCACCTCCAAGTGCAGGGATTACAGGCAT
GTGCTGCCAAGCCTATTCAATGCAGGTTTGGGGCTTGAACCCAGGGCTCTGTGCATG
CAAGCTAGGCACTCTGCCAACAGTGCCATAGCCCCAACTCAAGGCAAATTCTTGAGG
AAACCACAGATAGAATGGGAGAGTTATGGGATTGCAGACTCAGCTTAAAATACATC
ACAAAGTTAGGTTGTGTTGAAGCACTTGAATGTTTGTTTATATAACGATTCTATTTTA
TCATAACTCGGTCATCACAAGTTTACAAGGCAAACATTCTTAGTCCAGATAAGGAAA
CCATTCTAGAGGTCAAATGATTCCAGAGATTNACAGGGTATACGACAATANATTGGC
CCTGGCCNCTAATCAATGGCTGCTTCTTGCCGGGTAAAGAAAACATCCAATATAANC
CACNNCTTTCANAGCAANAATTTCAAAGACAACAAGCAGGGCAAAACCAGGGTCCA
AAGCAACCACT-3' and 5'-TGGGCGGGAAAGCAG (SEQ ID NO:35)
TTTGTCTTGTTGNTGAATTATGTTANNAAGCAAATGAAGTTATCTTCCAACACATGTG
AGGGAGTCCATTGTCTGGAGTCAAGCANTATTTCCCAACAGTTCTCTGTCAGTACAT
AACGCAAGGTCCTCCTTCAGTCAGAGATTTAAGACAACACTAAAGAGATGGAGAGA
AATAACACATCTGTGGTGTGTCAGGGACGCTGGCAATGGGCTGATCTTTTCCCATTC
NTTNTAAACTGGCTGTCCCAAAGGGCCCNTTGTATTTAGTCAAGTGACCATTCCAAG
CGCCAGAATGACCAGTGGAGGTGCAGAGAGCNTAGGGTGTCTTGGGGTCGCTGTGA
GGTGGGTCCCCTGCAGGATGTCTATGCACTTGCAGGCTTATACACCTGTGTCCCGCG
TNTTACTTGCCTCCTTCCACCCCTCTTAGGATACCTTCGCCGACAGCTCTGCTCTGCC
CGTGGTGACCATCTTTTGCGCTCCATTCTCTTGCCCTTTGTCTTCCCCTGGCAGCCTTG
TGTGACCCGCCTTTGTCCCTCCCTTCCTCTCCAGGACAAGCACGCAGAGGAGGTGCG
GAAAAACAAGGAGCTGAAGGAAGAGGCCTCCAGGTAAAGCCCAGAGGCCAAGGAA
GTTTCCAGGACAGCCGGACAGCTCCCGCAGCAACCTGGTTCCAGCAGCATCGGCTGC
TGGCTGCTCTCCCAGCACTGGGGTTCGGGGGGAGGGGGGTGGCCAAAGGGGCGTTT
CCTCTGCTTTTGGTGTTTGTACATGTAAAAGATTGACCTGTGA-3'.
[0183] In addition, the R010 clone contains an ORF from basepair 80
through basepair 727. This ORF encodes a polypeptide of 216 amino
acid residues. The translational start site was assigned to the
first methionine residue in the ORF. The amino acid sequence of the
R010 polypeptide is as follows:
33 MTLAAYKEKMKELPLVSLFCSCFLSDPLNKSSYKYEGWCGRQCRR (SEQ ID NO:36)
KGQSQRKGSADWRERREQADTVDLNWCVISDMEVIELNKCTSGQSFEVILKPPSFDGVP
EFNASLPRRRDPSLEEIQKKLEAAEERRKYQEAELLKHLAEKREHEREVIQKAIEEN- NNFI
KMAKEKLAQKMESNK ENREAHLAAMLERLQEKDKHAEEVRKNKELKEEASR.
[0184] The R010 clone was found to have homology to the stathmin
family of polypeptide, including stathmin, SCG10, and XB-3. In
addition, the R010 polypeptide was found to contain a unique 27
amino acid sequence (encoded by exon 3) that is alternatively
spliced to lead to the formation of two distinct mRNA
transcripts.
[0185] Northern blot analysis using a sequence from the R010 clone
revealed that the expression of L119 mRNA was restricted to brain.
In addition, R010 expression was found to be developmentally
regulated. Further, R010 expression was found to be rapidly induced
in vivo in the dentate gyrus in response to the multiple MECS
treatment and LTP stimulation, and rapidly induced in vitro by NGF
treatment of PC12 cells.
[0186] Another IEG nucleic acid clone was designated R042. The R042
clone is 3978 bp in length and has a nucleic acid sequence as
follows:
34 5'-CGGCGATGGCGGCGGCTGCT (SEQ ID NO:37)
GTGGTGGCAGCGACGGTCCCCGCGCAGTCGATGGGCGCGGACGGCGCGTCCTCCGT
GCACTGGTTCCGCAAAGGACTACGGCTCCACGACAACCCCGCGCTGTTAGCTGCCGT
GCGCGGGGCGCGCTGTGTGCGCTGCGTCTACATCCTCGACCCGTGGTTCGCGGCCTC
CTCGTCAGTGGGCATCAACCGATGGAGGTTCCTACTGCAGTCTCTAGAAGATCTGGA
CACAAGCTTAAGAAAGCTGAATTCCCGTCTGTTTGTAGTCCGGGGTCAGCCAGCTGA
TGTGTTCCCAAGGCTTTTCAAGGAATGGGGGGTGACCCGCTTGACCTTTGAATATGA
CTCCGAACCCTTTGGGAAAGAACGGGATGCAGCCATTATGAAGATGGCCAAGGAGG
CGGGTGTGGAGGTGGTGACTGAGAACTCTCACACCCTTTATGACTTAGACAGAATCA
TCGAACTGAATGGGCAGAAACCACCCCTTACCTACAAGCGCTTTCAGGCTCTCATCA
GCCGTATGGAGCTGCCCAAGAAGCCAGTGGGGGCTGTGAGCAGCCAGCATATGGAG
AACTGCAGAGCTGAGATCCAGGAGAACCATGATGACACCTATGGCGTGCCTTCCTTA
GAGGAACTGGGATTCCCCACAGAAGGACTTGGCCCAGCTGTTTGGCAAGGAGGAGA
GACAGAAGCTCTGGCCCGCCTGGATAAGCACTTGGAACGGAAGGCCTGGGTTGCCA
ACTATGAGAGACCTCGGATGAATGCCANTTCCTTGCTGGCCAGCCCCACAGGCCTCA
GCCCCTACCTGCGCTTTGGCTGCCTCTCCTGCCGCCTCTTCTACTACCGCCTGTGGGA
CTTGTACAGAAAGGTGAAGAGGAACAGCACACCCCCCCTCTCCTTATTTGGACAACT
CCTATGGCGAGAATTCTTCTATACAGCGGCCACCAACAACCCCAGGTTTGACCGAAT
GGAGGGGAAAACCCCATCTGCATCCAGATCCCCTGGGACCGCAACCCCGAAGCCCTGG
CCAAGTGGGCCGAGGGCAAGACAGGCTTCCCTTGGATTGACGCCATCATGACCCAA
CTGAGGCAGGAGGGCTGGATCCACCACCTGGCCCGGCACGCTGTGGCCTGCTTCCTC
ACCCGAGGGGACCTCTGGGTCAGCTGGGAGAGCGGGGTCCGGGTATTTGATGAGTT
GCTCCTGGATGCAGATTTCAGCGTGAATGCAGGCAGCTGGATGTGGCTGTCCTGCAG
TGCTTTCTTCCAACAGTTCTTCCACTGCTACTGCCCTGTGGGCTTTGGCCGACGCACG
GACCCCAGTGGGGACTACATCCGGCGATACCTGCCCAAACTGAAAGGCTTCCCCTCT
CGATATATCTATGAGCCCTGGAATGCTCCCGAGTCGGTTCAGAAGGCCGCTAAGTGC
ATCATTGGCGTGGACTACCCACGGCCCATCGTCAACCACGCAGAGACTAGTCGGCTC
AACATTGAGCGGATGAAGCAGATCTACCAACAGCTGTCACGATACCGGGGGCTCTG
TCTGTTGGCATCTGTCCCTTCCTGTGTAGAAGACCTCAGTCACCCTGTGGCAGAGCCT
GGTTCTAGCCAGGCTGGGAGCATCAGCAACACAGGCCCCAGACCACTGTCCAGTGG
CCCAGCCTCCCCCAAACGCAAGCTGGAAGCAGCTGAGGAACCTCCAGGTGAAGAAC
TGAGCAAGCGGGCTAGAGTGACAGTGACTCAGATGCCTGCCCAGGAGCCACCAAGC
AAGGACTCCTGAGACTGGAGAGCCATTGCTCCGTGAGCAAAGCCCAGGTGCCTGAG
CTGCCATGGCCACAGAGAAGACATGGAACCTACAGAGAAGACAGTCACCAACAGAC
AGAGCGAGCGACTGTGTGTGTGCAGAGGGAGGTGTGGTGTGCCGTTTGCGTGTGCAT
GCATCTGTTTACACTCTCATGATCCTGAATGTTGCCTGTGCTGGAGGAGCCCCTAGAT
CATGCCTTCTTACCAGGGCTGTTTCTTGACTTCCAGACATAAGACTAGAACCCGCAG
CAGTAACCGTCAGCCCAAATCTGCCCCTGGGAGCCCCAATAGGGTGGTAAGACCCT
AGCTTGAATTCTGGTCTCTGCCTCCCCAGACTCTTCTTCCTCCCTCCTTTTAACAAGG
AGCTGGAGGGCCACATTTTTGACTCTCATCTAAAGCATGGAGTTTCAGAGGCAGTCA
GAGTCCTGCTGACTTAGTTCCCACTTTTCTGACACTAGAACCTGAGCAGGCTGGAAT
AGATGTGTCCTGTTGATCTTAAACAGCCTGGCCAGTCTTCTTATAAAATCCTGTGCCA
TTAACAGGCTTCCCTGATGTCTAAGGCTACAGACTAGTGTGTTGTGTGCCCAGTACT
GCTTATGTCAGCCTCAGACATAATATCAGTCTTTGTAGAACCTTCTAAAAAAAACCA
CATGGGGAATAGACTCCCAGTCTTCTGTCCCTTCCCTAGCAGCTAAGGTCCAGTCTC
GACCTTCTAGAAGCTGTGGACAGGCTAGGGTCTGAACTGGTGAAAGAAACCCAGGT
CCCACAGCTGCAGGGCCCCTGGTTCCTCTGGCTGTACTCCTGACACCACATGCTCCA
GCCAGTACTGCTGATATCCAGCCAGGCAAGCTGGACAGCCTGGCTGGTCAGCACCTG
CCCTGCAGTGTCAGCTGCCCAGGACTGAGCTTCCGGAGACTCAGACAGACTTAGGG
GTGGAGCACTGCCTCTGGCAGTTGGCGAGAGGTCAGAGACCATGCCTGGCACATCA
ACATCTTCGCAGAGCAGCAGTGAAGGATTGACATAGAGAAGTCAAGCCTTGCTTTCC
AGGGGAGCCAACTCTCCCTCCCACTGTTGGGTCATATGGAGAAAGAAGTTATGAAA
GGATCTGGGGGTACCTGAGCAAGTCTTCCTTCCACCCCGTGGCCTGCATTTGAGCCA
CAGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGT
GTGTGTGTAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAG
AGAGAGAGAGAGTTTGTTTCTGTTTGGATTTTTGTTCTCACATGTAACATTAAGCTGG
CCTCTGGGCCTTTTCCTCTCTACCTCCCCTGTGACCTTTCCTAGCCTCAGAGTTGTTAA
TGCCCTTGGCCCTGGCCTTTTTTTGTGTCAGACCAGAACCCTGGGGTCAGGCTCCCC
CCTCCAGCTGTCTAGCACATCTGACAGGCTTCTTTTTGAGATGGCCTCAGGTTTTCTC
AGCAGAGAGCTGCCTTTAGTCCAACTGTTTATGTTCATCATCCTGACTAGAAGCATC
CTACGATTGTGTGAAGAAACGGCATCTGTGATGCCATGTTCAGAGTCATGGGGTGTG
GCCTCCCTGTCCCTAGCCCCAGGCCAAGAGGAAAGGGCCAAAGGCTCTTGCTGGAG
GGACAGTAGAATGCGTCTGGAGAACTGGTCCCAGAGGAGCAAAGGCTTATTCTGGG
GCCAGTATTTATTTTGCAACATCTTCAGCTATGGGGACAATGGCCTTCTCTGCTTTTT
TGATGATGGCTCTCTCCTCAAGGTACAAGTTGGCAAGGTCATCTGTCCTTCCACCTCC
TTGACATGTTGGCCCATTTCCAGGACAGCCTTCCAGTGAATGGAGCAGACTATTCCA
CAGCTGTGGGATAGAGTGTCTTGGAGCCCTGGAATGACTTCATGCCTCCTTTTGCCT
AGCCTGAGTGGCCCTGAGGACTGTCACAGAACAGTGCCCCATGTCCTGCTCCTGGGC
CCGAGCATGGGGAAGAGATGGTTGCAGGCAAGAGCACTTTACAGCATTCCCCATTG
CTGGGAAGGTTGTTTCTCCTACAGTGTGTGAATACTTACCTGTTTTATAAATGTCTGA
TCCTGTCTGAGTAAAAAAAAAAAAAAAAAAAAAAA-3'.
[0187] In addition, the R042 clone contains an ORF from basepair 51
through basepair 1790. This ORF encodes a polypeptide of 580 amino
acid residues. The amino acid sequence of the R042 polypeptide is
as follows:
35 MGADGASSVHWFRKGLRLHDNPALLAAVRGARCVRCVY (SEQ ID NO:38)
ILDPWFAASSSVGINRWRFLLQSLEDLDTSLRKLNSRLFVVRGQPADVFPRLFKEWGVTR
LTFEYDSEPFGKERDAAIMKMAKEAGVEVVTENSHTLYDLDRIIELNGQKPPLTYKRFQ
ALISRMELPKKPVGAVSSQHMENCRAEIQENHDDTYGVPSLEELGFPTEGLGPAVWQGG
ETEALARLDKHLERKAWVANYERPRMNANSLLASPTGLSPYLRFGCLSCRLFYYRLWD
LYRKVKRNSTPPLSLFGQLLWREFFYTAATNNPRFDRMEGNPICIQIPWDRNPEALA- KW
AEGKTGFPWIDAIMTQLRQEGWIHHLARHAVACFLTRGDLWVSWESGVRVFDELL- LDA
DFSVNAGSWMWLSCSAFFQQFFHCYCPVGFGRRTDPSGDYIRRYLPKLKGFPSR- YIYEP
WNAPESVQKAAKCIIGVDYPRPIVNHAETSRLNIERMKQIYQQLSRYRGLCL- LASVPSCV
EDLSHPVAEPGSSQAGSISNTGPRPLSSGPASPKRKLEAAEEPPGEELS- KRARVTVTQMPA
QEPPSKDS.
[0188] The R042 clone was found to be a photolyase receptor based
on sequence alignment data. In fact, the R042 clone was found to be
the rat paralog of human and mouse clones based on the following
observation. The identity between the human and the mouse clones is
considerably higher (97%) than between either the human clone and
R042 (72%) or the mouse clone and R042 (71%). This lack of a higher
identity between the mouse clone and the rat R042 clone is more
than that expected from species-to-species differences. Thus, the
R042 clone most likely is a different member of the family of
photolyase/blue-light receptor homologues. The translational start
site was assigned to the second methionine residue from the 5' end
based on the alignment data using the human and mouse members of
the photolyase/blue-light receptor family.
[0189] The R042 clone potentially has two differentially spliced
forms at the 3'-end. The difference between these two forms is 142
bp. The shorter form was found in four clones while the longer form
was found in one clone.
[0190] Northern blot analysis using a sequence from the R042 clone
revealed that the expression of the R042 mRNA was strongly
upregulated in response to the multiple MECS treatment.
[0191] Another IEG nucleic acid clone was designated R053. The
primary library screen produced 40 positive signals that were
isolated. The following nucleic acid sequence is within the R053
clone:
36 5'-TTGGCACACAAGTCTGTCTTCAGGACAGCTGATCCATTTTACTTA (SEQ ID NO:39)
CRAATTCAGAAAGTAAACATTGGCAGTATGGATCTGGTTACTTCATGGTAACTGCTC
TAGAATTTACGCCAAGGCCATCTCTTTTGCCTCACTGTTTAGTGACCGGAGTAAAGC
ATGGGGCCACTGAAACTCCACTTTACAATTGGGCTTCTAAATTTAAGGAAAAATTTT
TTGATTTAACCACAACTGGATTCCAAAGTTCATCTTATTCYAAATTAGGCCCACTGA
GCCTGTGATGTTTTGGAATATATGATTAGTCCACTTGGTTCACTGGATGTTACCTAT- C
ATGTTATGTAGAGAAACAGCCATAACTATTGGTCACGATGTCGTCCTCCGAATTGG- G
AATGGCTCTGTTGTTGGAAACAAAGTATTTGTAAACACGTTGATCAAAGCGGTGTG- C
TTTGGCCTTTCCGGGAATCACTGATTATGTTTGAAAACTTCCTTTAATTGTATTTG- CA
ATAAGCTATTNTCCCTTNTNATGNCNCTGCCATGCTTCCTTGCTTTGCACTGTGG- TCG
CATGCCATCNGCTGGTTAACCCANGATGGCTTGCTGCNCTGATATNCACCATGC- NAA
ATACCACTTCT-3'.
[0192] Northern blot analysis using a sequence from the R053 clone
revealed the presence of a 4.9 kb mRNA transcript. In addition,
this analysis revealed that the expression of the R053 mRNA was
marginally upregulated in response to the multiple MECS
treatment.
[0193] Another IEG nucleic acid clone was designated R055. The
first library screen produced a clone designated R055-7 having a
1.7 kb fragment. A second library screening using the 5'-end of the
R055-7 as a probe produced several additional clones having
fragments of about 3.0 kb. The following nucleic acid sequence is
within the R055 clone:
37 5'-TGAATTGCAGTAACT (SEQ ID NO:40)
AGCCTTGCCTTTCTATTCTGTAGAAATGACAGGGTCTTCACAATCCTTCACCAGTGGC
TACTAAGCTATAATTAGCTGAATAGAAAGAATGTGGAAGTGGTCTGAGGCATATAG
AGCATATGCCAAGAACACTACCATATATGGCATCAGCTTTGGTTACCAGAGAAATTT
TCTTAGTCATTAGACCATATAACAGTAATATATCATATGTAAATCTTTAGATTTCAAT
TTGAGAATCCTCCAAAAAAAAGGAGCAAAGAATGCATAAGCTATGTGTTGGCAAAA
GTAATTTATATTAAAATTTTGACCTGCCTTTGTAAGATTAAGTGGTAAATGTCATAGT
GGTGGGTTTTTACGTCTTAACCAATCTCTGAGGTTTATTTCTCCTGCAGGGGATGGTT
CATGGCCTCTCTTCCCGCTGTAGGAAGATAGCAGAAGGATGAGGATTAATTGTAGCA
TTTCACTGATCCTCGTCCCAGGGACTAGGGACAATAGAAATCTGCAAACATGGAGA
GTCTGTCATAAATATTTGCTTTTTGAAGGTGTTGGTCTTTGTTGATTTCTGTCAGAAA
ATGGCATTATACAAATTATGGGGAGCAACCAACTTTTCTGTTCTGTTTTTGAAGTGCT
ACTATGAACCATTCAGAGTCGTATTTTTTTTTTTTAAAATTTTGGCCAGATATCCCCA
GCTAATGAAAAATAG:TCACCATTCCTTGAAAAAGTTGGAAGCTAGAACCCCCAATT
CCAAATTATTGTTGAAGATGTTTCTCAGGCTACTGTATATAGAAATAATGTTTTTAAG
AAAAATCAAAGAGAGGAGAAAAAAAAAACCTATGCAGAGACCCTACTACTTTGTGG
TTTCTATTGTCCCTATACATCATTTCAGCAAATCTACTGGCAGTTCTTGTCAGCAAGT
CCTTCAGTGCATATGCTGCACAAAACAAAACAAAAATCTGCATGGCACCAAAAACC
AAACAAGCAAACCAAAAACCCAGACACCCTATGTATCTGTTGGAGGCATGTAGGTG
GTACAAATGACTAGCCATGAGCACACATGGCTTCTTGTCATGTCACTTTTCATAATTA
TTTACTGCAAAATGATTGAGAGGCTTTTGGTGCAGGCAGCCATTAGCCTGCTTCCTTT
GTTACCTCTGGATCACTTTGCAGTAAATTGCAGGTCTTTTAAAAGATTCAAGCTTCGG
TTTTCTCAAAACAAAACAATTATCCTGTCTTACCTGAAAATGCAGGGTTGTGGGCAA
AAGAGGCTGGTTATAATAATGCCCTCATATTGAGTGGTCTGTAAATGGCTGCACACT
TCAGGCACTAGAGTTGCCGAGGATGCGTTGTTAATGTGACCTTGACTGGCTTTACAG
GGGTGTAGAACAGTCTACACGGGCGACTATTTGCATCCATCTTGCTCTCGAGGTGGA
TGGAAATAAGAAAAGGCTGGAGTGTGTAAGTCATGCACATAAGTATTCACTGTAAA
TTTTATTTTCATTTTTAACCCAATTATGGTACTTTGTCCAATGCACAACTGATCTCTCA
GTAGATATTCATTTGAAAATAGTGTGGCCTTGACCAGCGAGAAGGGGAAGAAGTGA
CTTAGCTTGTGTTAAGATGACCTGTTTGCTGAGAGTGGTCATTCTGCAGCACCCTAAT
GTCATGGTTTTGATTAGGGAGAGTTAATGTTTTTGACCCTGAATTGAGTTTTCTTCTA
TTTTTAGGAAGTATCAGAATTGCTCTGATGAGTAACAAAGTTGACTGTTTTGATGTCC
AATCTCAGGTTTTAAAATAGAGTGGTATAAAAGTCCACTGTTACTAATTCTTAAGAC
AATTTTGATTTAGTGTGCCCTAAAAGTCACGTGCATAATAAGGCCTGCTCAGAGGGC
AGGGCCTCCATCTGTTTGCTCCTTTCCATGTTGTACGCACTTCACTTGAAAAGGTGTC
AAGTGACTTTGCATTGTAGATTTCCATTTTAACCCCAACATAGTTCTCAAAGATAAA
GCACTTTTTGAACATGAAATACATGGGTAATGTGTGATGTGGATCATGGTTTCTCAG
GCCCCTAGATAATCCACTTCTGAGTATTGTTCTATGTAAGGAGAATAGAGGTCTTCG
CTAATGTTCGAGTTTGTATTCCTGAATGGAATGCACTTGCTAGTTTCCAATGGATGGG
AGAGTAAACACTGCTGCATTCACAATTGATACGTTGCTTTCCCTTGAGCCTTAAGGT
AACTTTTCTTTTCTGTCAACAACAGCACTGAAGTTCTAGTAAGTGAATGAGATTATCT
GTTTTCAGGGTTGGTTTTAGAGTACTGTAAATTAATTAGCTGTCTTCCTAAAGAGGA
ACTCCCTTTAACTCCCTTCGATAGACTGAAAGTGGGTGTGGGGAGGGGGAGGGAAG
AGAGGGAGGTAGTTTGTAGAAAAAAAAAAAAAAAAAAAAAAAAA-3'.
[0194] Northern blot analysis using a sequence from the R055 clone
revealed the presence of a 7.3 kb mRNA transcript. In addition,
this analysis revealed that the expression of the R055 mRNA was
marginally upregulated in response to the multiple MECS
treatment.
[0195] Another IEG nucleic acid clone was designated R061. The
following nucleic acid sequence is within the R061 clone:
38 5'-GGCCCCCCCTANAAGGTCGAGGNTATCGATAAGC (SEQ ID NO:41)
TTNAATATCGAATTCGGCACGAGGCCACCAGGTCTTTGCATTGTCTCTTTAAAAGTG
GTGTATAAGGGGGAAATTGGCAAGACAGACATTTCTAAACAGAGGGGAACACAGAC
AGACAGACAGACAGACAGACACACAAACACACAAACACACACACACACACACACA
CACACACACACACACACACACACACACACACACACACACACACCCCAGACTGCGTA
TGTGGCATAACATACAGCTTGCATGGGAAGCAGCCCCCTGCRCATTGCTTATACATC
CTCGAGTCCTTTCATCTTTTTTCCTAAAACGTGTGCACCCGCTATAAAGTGGGTGATG
GGCTCGTCAGAGCTGGGCTGATTCTGTGGCCGGTGACCACCATGCCTCAGGTCCCTC
AACCTCCATCACCCATGGCCCAATCCATAACTGCCACCCTTGAAAACCCAAAGCAGT
CTGAGGGTGCTCTCTGCCTGTCACTCAGAGGCCTGGGACGTTGAACCCAAAAAAGCT
AAACTTATGAAAGCCGGGCTGAAATGGGGCCCGGGGCCTGGGATAGCTCAGGCAGG
GGTTTTCCACTCTGATGTTTCCACTGGGCCAGTTTTGTTTCTTTGTCTCTATTTTCTCT
GTTCATCCCGCTGAGTGTTTGTATCCATGATGATTCCAGCATGAAGTACGTAGCACA
CTCCAGTTAGGAGAAATTTTTTAAAGATACAAGACTAGCGTGGTGGTGAGATGAGAT
AGTCTTCTCGTGCTCGCAGCAACCTGAAGGGGCAATAAGGACAAAGAAGGCCATGT
GGCAGGGTTAGCCCCCTCCAGACCAGGGGTACAACGGACAGTTGTGGTGAGCCTCG
GAAAGGCAGGGGTAACCTTCCCTCTCCGTTCTTTCACCCATGGCCAGAGCAAGGCAGG
TAGTGAAAGGGATATGCTTGATGCAGAAAAGCCAGCTCAGGCATGGCAGGTGGGAT
TTATAGCTGGTTTTGTTTAAAGCGAAGGCCTGATATTTGATAAATGCAGTAACCAGC
GGTTGAGAGTGACAAGCCCTTAAATGCGAACATTAATCAAAGGAGAACTTAAACGG
CCCCCTTTACAGAAGGACTT-3'.
[0196] Northern blot analysis using a sequence from the R061 clone
revealed the presence of a 4.9-5.0 kb mRNA transcript. In addition,
this analysis revealed that the expression of the R061 mRNA was
marginally upregulated in response to the multiple MECS
treatment.
[0197] Another IEG nucleic acid clone was designated R066. The
following nucleic acid sequence is within the R066 clone:
39 5'-CGAGTTTTTTTTTTTTATGTACTTTGAAAATATAT (SEQ ID NO:42)
TTAAAAACATTAAAAATTCTATATTTAAAACATATATTATATGTTAATTGGTACACTT
AAATAGAACCTGTATTTACAATAGGCTTCTGATGTGGTTAAGTTTTAATGCCAATTTT
TTTTTCAATAACATAATTATATAAATATACTAAAATACAATAAATATTTTTCTTGTTT
TACATGGTGAATAATATCTTTACCATAGAGAGAACAAGGCCACAGACATTTACTTAC
AGTTTCAATGGGAATCACTATAAAAAGCATCAGGCCTGCTGCCATGCATGAAACACT
TCTGCCAAAAAGAGACCACAGCAAGACTTTCAGAACAGAACAGAACAGAACAGGAC
GGAAACAGAACGAACAGAAACAGAGGAGAGATTTTAACAAATCAATCTCAGGTCAA
CATAAACCACCGACATGGAGCTATGATGTATCTTAGTGGGTATGAGAGCCAGCCACT
GACCACACAGTTGCGGAGGGTCTCCTATGAAGCCACCTAATCGACCTGGCCCTTCGA
ATACCGTGAGATTGTGATGGGGCTCCTTTTATTTGTTTGACTAACGTCTCTCAGAATG
AAGCTGCAAAAAGTTAGCATATAGCAGATATTCAAAGCATTCCTTAATAGGTTAAAA
ATGATGACAGAGATTAATGTTGTCAAACGGCACAAAACAATCTAGGCTACGTGAAG
TCTTCCAAAAACAGGGGATTCAGTGGGACTCCAGAAGACAGACTAGTTCTAAAGGA
ACAGTTGAACAAAAAGAAACTATTTGCTGATGGTATCTTCACTCCCTGAGTCACAGT
GGACAGCCACTTTGTTTCACCCTTTCCACTCCTAAGATGAAGCAATTGTTTGCCTCTT
TTTCTGATGCCCAGGAGCCCAGTCAGGTAACCACTAACACATTCGCGCTGGCGGAAA
ACCTCACTAGGGAAATGGGCTTAACACTAGTTCTCATTGGGGCCATTCATTCAGGCT
TCCAGCTTGACTTCTCCTAACCCCAAGAGGTAAAGTGTAGAAGGGACCCTTGTGCTG
AATGGACAGAACTATCAGGAGCTTTCTGTGCTCTTCACTTAAGCAGTATTTCCTCCTG
TGTTCTTGTCTTTCACAGTGAAAGCACCTTCCTATGCCTTGTCATTCTAGCCCTTAC
AGACAGACATTGCTCATTCTGCCTAAGTTTTGGTGCTTTTTCTGGTTTTGTTTGTTTGT
TTTCTTCTTTCTTTTTTCCTTTCACCAAAATGTCTCAAAAAAATAAATAAATAAAACC
TAGGCTTCCTGAAGTCTAAGCGCAAAGAAAGTTAAGTCTCTTCACAGCAAACATTTC
CCATCATGCTGCACTGATAGCATCACTGCTATGCCATATTTGGATCCAAAGCTGCTC
CAGGTTAATCCAACTTTATCCATAATTATTTAAAAGGGATGGAGGCCATAAATGGA
TTTGAG-3'.
[0198] This clone is similar to BDNF.
[0199] Another IEG nucleic acid clone was designated R089. The
first library screen produced a clone having an insert of 0.5 kb. A
primary screen with a portion of this clone produced seven positive
signals that were isolated. The following nucleic acid sequence is
within the R089 clone:
40 5'-AGTCTGGGACTAAAACGTCACAGCAGAAAAAAAATAAAAAAAAAT (SEQ ID NO:43)
AATTTGCTTTTTCTTTCTTTCATTTAGCAGCATAAATAAGTTTGGCCACTGGGAGTA- C
AGTACAGGGGTGGGACAACGATCCCGTATTTGAAGACCTACTTCTAGCACCAGCAT- C
AAGAACTAAATCCACCTCAGGACTCACAGAACCCAGGACAACTTGCCATCTTTGAG- C
AACATATGCATTGAAGAGTGTATATAGAAGCAACAGTAAATAGATTAACAGAGGCT
AATACTGTGATTGATTGACATTGGCAATGGTTGGCAAAAAAAAAAAAAAAAAAAA- 3'.
[0200] A portion of R089 was found to be highly homologous to a
region within an EST from GenBank representing a cDNA clone from
ae87b04.s1 Stratagene human schizo brain S11 (accession #
AA774778).
[0201] Northern blot analysis using a sequence from the R089 clone
revealed the presence of a 3.8 kb mRNA transcript. In addition,
this analysis revealed that the expression of the R089 mRNA was
marginally upregulated in response to the multiple MECS
treatment.
[0202] Another IEG nucleic acid clone was designated R095. The
first library screen produced a clone having an insert of 2.0 kb. A
primary screen with a portion of this clone produced 53 positive
signals that were isolated. The following nucleic acid sequence is
within the R095 clone:
41 5'-ACTTGATAAAATTGTATTTTTTTTTCTACAGTCATTTGTACAATTTG (SEQ ID
NO:44) TTACAAAACCATAGAAGACTACAACTTGTTTTAAATCATTTTTGGTCTG- CAAATATGT
AAAATCTGTGGTGCAATTTATCATGTATTTACAGGGCCTTGTTAGTCA- TTTTCAATGAT
TATTTCAACAATGTCACACTCTCAACATAAGACATGGCTTAAGACA- AATATATTAGT
ACATANATATTCTGAGAACATATTTCCATNAATGGAAAGTNGCTGC- TAATACANATA
CAGAATATACATAAGNTGTTTTCTAGCTTTTTAAAACAGTTTTTAA- AATGGNAANGT
GAAAAAAGAGCCCCTAGGANCATTTTATCCCAAAAAAATCCTTACN- AAATATTNAA
GGGGCCAGGGGGGGAATTAAAAATCTAAAAANGGTGGTC-3'.
[0203] Northern blot analysis using a sequence from the R095 clone
revealed the presence of two mRNA transcripts: one 2.5 kb and the
other 3.2 kb. In addition, this analysis revealed that the
expression of the R095 mRNA was extremely strongly upregulated in
response to the multiple MECS treatment.
[0204] Another IEG nucleic acid clone was designated R113. The
following two nucleic acid sequences are within the R113 clone:
42 5'-AARGGGRCCACCCCACCGSGCTA (SEQ ID NO:45)
AAGGCCCAGGGGCCCCCCCCTTGGAGMCCCAGGGGTTTTGGCCCMCCCCCTCACCC
AAATGGTCTGCCAATGACCCAGGTACTCACAACATGTTCCAGGAGGAGMCTGGGGC
CAGGATTTTGACCAGAGGGTATGGGAAGGGAAAGGGGAGAAGAAATCGACATTTAT
TTTTATTATTTATTTTAAATGTTTACAWTTTCTTTGTGTTGTTCCAAGCCCTGAATAG
AAACAGATAGCATTAAAGGACTCTGTTCCCACCCCTTCTCTGTCTCTCTCTCCCCCAC
TTGTGCTAACTTAGGATAACACTCTCTATTTCGTTTTGTTTCTAAAGTGATTTGTGGA
CTTGTGCCGTGTGAACTGCATTAAAAAGGTTCTGTTTTCAAAGATCGATTGTCGTTCC
TGTGGGGACAGTGGCTCCTAAGAAATCTGCATTGTAGGAGAAGACAATGAAAGACC
CTGGCCCTGTCTCTCAAAACTTAACTCTCTGTATGATTTAAAAAAAAATTCCATTTAC
TTTACTTTGTGGTTACTTGATTTTGAGGAAGAAAATATTCAACTTTGTATAAAGACTA
GGTATCAGGGTTTCTTTTGCAGTGGGAGTTGTATATATATCGTATTTTGGTATATCGT
AGAAACTCAAGCTTTATGCATCCGTATTTGGGATATGTCAATGACGTGCAGTGAAAT
TTGCTATTAGACCCTGGAGGCAAACGAGTTGTACAAGGTTTTATGGCTCCATGGGGA
ATTCTAATTTCCTTTCTGGGGACCTTTTGTCCCGTTTTTACAGTAATGGTGAAATGGT
CCTAGGAGGGTCTCTCTAGTCGAATTCTCCAGGCAGGACCACGTGCTCAAAAAATCT
TTGTATAGTTTTAAATTTTTGAGGAGTATCTCTGCTCAGAAGCATCTGTGGTGGTGTG
TGTTGCGTTGTTCTGTGTACTGTGTGTGACACAAGCCTACAGTATTTTGCACTAAGGA
AAGCTGTTTAGAGCTTGCTGCTATGGAGGGAAGAACATATTAAAACTTATTTTCCCT
CGGGGWTTTRTWCWMGTTTTATGTWCTTGTTGTCTTGTTGGCTTTCCTACTTTCCACT
GAGTAGCATTTTGTAGAATAAAATGAATTAAGATCAGMWRWRWRMAAAAAAAAA
AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA-3' and
5'-AATTCCCCATGGAGCCATAAAACCTTGTACAACTCGTTTGCCTC (SEQ ID NO:46)
CAGGGTCTAATAGCAAATTTCACTGCACGTCATTGACATATCCCAAA- TACGGATGCA
TAAAGCTTGAGTTTCTACGATATACCAAAATACGATATATATACAAC- TCCCACTGCA
AAAGAAACCCTGATACCTAGTCTTTATACAAAGTTGAATATTTTCTT- CCTCAAAATC
AAGTAACCACAAAGTAAAGTAAATGGAATTTTTTTTAAATCATACAG- AGAGTTAAGT
TTTGAGAGACAGGGCCAGGGTCTTCATTGTCTTCTCCTACAATGCAG- ATTTCTTAGG
AGCCACTGTCCCCACAGGAACGACAATCGATCTTTGAAAACAGAACC- TTTTAATGC
AGTTCACACGGCACAAGTCCACAAATCACTTTNGAAACAAAACGAAAT- AGAGAGTG
TTATCCTAAGTNAGCACAAGTGGGGGNGAGNGAGACAGAGAAGGGGTGG- GAACAG
AGTCCTTTAATGCNATCTGTTTCTATTCAGGCTTGGAACAACACAAAGAAA- TGTAAA
CATTTAGNATAAATAATAGAATAAATGTCGGGTTCTTCTCCCCTGTCCCTT- CCCATAC
CCNCTGGCAAAATCTGNCCCAGGTCCTCCCGGAACATGGTGNGAGTACCT- GGGTCCA
TTGNAGNCCATTTGGNGAGGGCGTGGCCAA-3'.
[0205] Northern blot analysis using a sequence from the R113 clone
revealed that the expression of the R113 mRNA was upregulated in
response to the multiple MECS treatment. Specifically, R113 mRNA
expression was induced seven fold by the multiple MECS treatment as
determined from Northern blot data using total RNA from rat
hippocampus (Table I). In developmental studies, the expression
level of R113 was found to be low and unchanged in embryonic as
well as post natal development.
[0206] Another IEG nucleic acid clone was designated R114. The R114
clone is 3318 bp in length and has a nucleic acid sequence as
follows:
43 5'-GGCACGAGCCGAGGCT (SEQ ID NO:47)
CAGCACAGCACGGATAGGGGCGCGGAGCGCACTGAGAACCCTACTTTCCCGTGAGC
CCGAGCCCGGCAAATGGGCGAATGAAGAAGGAGAGCAGGGACATGGACTGCTATCT
GCGTCGCCTCAAACAGGAGCTGATGTCCATGAAGGAGGTGGGGGATGGCTTACAGG
ATCAGATGAACTGCATGATGGGTGCACTTCAAGAACTGAAGCTCTTACAGGTGCAG
ACAGCATTGGAACAGCTGGAGATCTCTGGAGGCGCGCCCACCTTCAGCTGCCCTAAG
AGCTCACAGGAACAGACCGAGTGCCCTCGCTGGCAGGGTAGTGGAGGGCCTGCTGG
GCTTGCTGCCTGTCCCTCCTCCAGTCAACCATCTTTTGACGGCAGCCCCAAGTTTCCA
TGCCGTAGGAGTATCTGTGGGAAGGAGCTGGCTGTCCTTCCCAAGACCCAGATGCCA
GAGGACCAGAGCTGTACCCAACAAGGGATAGAGTGGGTGGAGCCAGATGACTGGAC
CTCCACGTTGATGTCACGGGGCAGAAATCGGCAGCCTCTGGTGTTGGGAGACAATGT
TTTCGCAGACCTGGTGGGCAACTGGCTAGACTTACCAGAACTGGAAAAGGGCGGGG
AGAGGGGTGAGACTGGGGGATCCGGTGAACCCAAAGGAGAAAAAGGTCAGTCCAG
AGAGCTGGGTCGTAAGTTTGCCCTAACTGCAAACATTTTTAGGAAGTTCTTGCGTAG
TGTGCGGCCTGACCGAGACCGGCTGCTCAAGGAGAAGCCTGGTGGATGACTCCTAT
GGTTTCTGAGTCACGAGCAGGACGCTCGAAGAAAGTCAAGAAGAGGAGCCTTTCTA
AGGGCTCGGGACGGTTCCCTTTTTCCAGCACAGGAGAGCCCAGACATATTGAAACCC
CGGCCACAAGCAGTCCCAAGGCTTTAGAACCCTCCTGTAGGGGCTTTGACATTAACA
CAGCTGTTTGGGTCTGAATTCGAGAGATGCTCACTGACCTAAAATGCAGACTTTGTGA
GGGCCCTGGGGGAGGGTGGGCAGATGGCATGGTCTTCAGGCCAGATGCAAGTTCCC
ATCCTCAGAAAGAAAGCAGAGTTCTTTAGTCAGGCCTCAGTAGAACAGTGGAGAGAG
GCTGTCACAGGCCAGGCTGAGCTGAGTCCCTGGAGAGAATGTGTGTATTTGTGTGTG
TGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTATGTGTGTGTGTATGCGTG
TGCATGCACTGTTGTTGTTAGAGGCTGGATGTGACAATAATTGGGAGAGGCAGGAA
AGGAGTCCAGGACAAGCCTATGATATTCCTCCATTACCTTACCCAAGACCTCATTTG
AACATTCTATATGCAAAGGGGCATTTAGCCCTCAGGTTTCCCAGAGGAACTCCCAAT
AAAGACCTGTCTCAGGGACCCCCAACCATTTTTTAATGGTCTGCTTCCCTGACAAGG
CACTGATGCAGGCAAGGGGTTTGTTTTTGTTTTAAGGGTTGGTATCCCAGAATGGAG
CACCGGAAATAGGAAAATCCCTATTTATAGCCCTTCCTAGGACCAAGATTTCACCCA
TGGCTGGGTGCTGGGGACGCAGAACAAGCAGAGGGGTGTGCGTGCGTGCGTGCGTG
CGTGCGTGCATGTGGTGTTGAGGAAGCCTGAGATGCTCCCAGATCTCTAAAGTGCAG
AGGAGAAGCAATGTGCGTTCACCCCGGTGATTCCATAAGCAGCCATCTCTGAGAGC
ACACTCGGCTGCCAGGAGGAAAAACAGGTCAGGCCAATCTCATGGTTATCAATGGA
CCCTAGAGTCATACGCTGCCTGGTCCAGCAGTGAGAGCCCATCCTGACTCCCTGTTG
CCTATCTTAATGCTCCTGCAGGGCAGCAGATGGTTGGGGTGAACCCAGAGATAATAC
CCATACATTGAGAACATTTCTTAGTCTACATCTCATAGTCATTCAGCGAACTGGACA
CATCTACCCGCATCACCCTGGAGGTCAACAGGGGACCCTGAGGGTGGGGCTGATGC
CAGGCACTTTATATAGTGAGCAGGCGTGCAAGTCTGGGACCCAGGGAATCCATCTCA
GCCCCCACCCCTTAGCCAGGAGAGAACAAAGTAGGCCCCTGTTCAAGCCCAGCTCG
GAGGCTGCCTTAGCTCCTCCTTCGCCCCCTCCTGCAGACCCAGCTCAGCTTGATGAG
GTGTGACAACTGCAATTAGAGGCAAGCCGCCTGCTGCCCCCAGAGCATTAAGAGCA
AATTAGAGAAGAAAAATCACAAGAGAAGCTCTTCTGCCTGCAGTCTAGACTCCCAG
GGGACTGGGTGGAGGAAGGAAGAGCTTAGGGCATAGGGATGAGGAGGTAAAAGTA
ACAGCAGGAAGGGTCACCTGCAAGTTCCCACGCAGTTAAATGATAGGTGGCCTTTTT
TTTTTTTTTTTAATCTGTAGCTTTTTGTCAGGCAATGTGCCTATCTCTTTCAGAACAAT
TAATCAGTGGGGTCAAAGGGCCCTGCCATGCTGGCTGCCCCCATCAGGCTACTCAAA
AAGGAAAGCAGTTCCAAGCTCCAGCCTGTGGGCATCAGGCCTATCTGCTCTGGCCTG
GTGTTTATCAGCTAGGCTCGCTCTTTCTGGTCAAATGGGTCCTCATCCATTCTGTCCC
CACTGAACTTCTGTCTCTGGTGAAGGAAGGTAACTGTAGCTGCCTCTGATGGCTGCT
GCAATGTGTGTGGAGAATGAACATGTGAAAACCCCACACCCTGAAGGGTGGCACAT
ATGACACATTTACTCAAGAGGACACAGGACTGGGACGGTGTAGGAAGCCAACTCAT
TTGTTTTGTGGACTAGTCACTGTTCACATTATTTAAATCGACTGACGTGACAGACTCC
TTCTTTGACTGGGCACTGTGACAGAAGGAGAGAACTCAGCAATGGGAAAGCTGGCC
TCCACAGCTACCAAGGCACACAAAGAAATCCAGTTAACCACCACCTGGCCAGAAAA
GGGTCAAGGGACCAAAACAAAATGATTAGCAAGTAATTTTGGCTTCTAAGAGAACC
CACAGGTGTCTGTCACCTTGATCTTTATTTTTCTGCTACACCCAGGAAATGGTTGCTC
ATTTTACCCAGTAGACTCGGAGAAGTTAATGCTTTCAAGGTCACACAGTACAAAGCT
GGGATTGAAACAGTTTGTAACTGACTTCCAATCTTGTGTTCATGCTACCTGGCAAAC
TGTCCATATTTGCTCCACAGCCAGATCCAGAATAACATTTGTCTCCTCTCGTGCAAAA
AAAAAAAAAAAAAAA-3'.
[0207] In addition, the R114 clone contains an ORF from basepair 94
through basepair 993. This ORF encodes a polypeptide of 300 amino
acid residues. The translational start site was assigned to the
first methionine residue in the ORF. The amino acid sequence of the
R114 polypeptide is as follows:
44 MKKESRDMDCYLRRLK (SEQ ID NO:48)
QELMSMKEVGDGLQDQMNCMMGALQELKLLQVQTALEQLEISGGAPTFSCPKSSQEQT
ECPRWQGSGGPAGLAACPSSSQPSFDGSPKFPCRRSICGKELAVLPKTQMPEDQSCTQQG
IEWVEPDDWTSTLMSRGRNRQPLVLGDNVFADLVGNWLDLPELEKGGERGETGGSGEP
KGEKGQSRELGRKFALTANIFRKFLRSVRPDRDRLLKEKPGWMTPMVSESRAGRSKKV
KKRSLSKGSGRFPFSSTGEPRHIETPATSSPKALEPSCRGFDINTAVWV.
[0208] A portion of R114 from base position 111 to position 210 was
found to have 98 percent identity with the mouse G protein-coupled
receptor EBI 1 (accession #L31580). This homology, however, ends
with position 210. In addition, the 100 bp region of 98 percent
identity in the EBI 1 clone appears to be an artifact produced
while PCR cloning EBI 1. This "identity" region in R114, however,
is not an artifact, since RT-PCR with primers located in the 3'
untranslated region of R114 and the middle of the "identity" region
(139-164 bp) was used to obtain portions of the R114 clone. In
addition, a portion of R114 from base position 143 to 601 was found
to have very strong homology with a human EST obtained from
prostate tumor (accession # AA595469). This indicates that the
entire "identity" region is from one gene and not a product of
concatamerization of the R114 clone and EPI 1.
[0209] The alignment of the human EST obtained from prostate tumor
with R114 revealed a very high level of identity at the 5' and 3'
ends of the overlapping region and a somewhat lower homology in the
middle. In addition, 13 base insertions and deletions were
identified between the EST sequence and R114. After excluding 7 of
the 13 differences because they would have caused a frame shift,
the two sequences were translated and compared. This comparison
revealed an 81% homology at the nucleic acid level and an 85%
homology at the amino acid level. Interestingly, no homology was
found between the two sequences before position 143 of R114.
Position 143 is six bp before the third methionine residue. Thus,
the translational start site of R114 may be the third methionine
residue in the ORF.
[0210] Further, 95% homology was found to exist at the nucleic acid
level (98% at the amino acid level; there is a one base deletion in
the EST that is probably an error of sequencing) between the 3' end
of the R114 ORF from position 580 to about 987 and the full length
of an EST from mouse mammary gland (accession # AA472513).
[0211] Northern blot analysis using a sequence from the R114 clone
revealed that the expression of the R114 mRNA was moderately
upregulated in response to the multiple MECS treatment.
[0212] Another IEG nucleic acid clone was designated R198. The
following two nucleic acid sequences are within the R198 clone:
45 5'-TTTKTTTKTAATTTTTTTTTTTTNATTTGGGTTGA (SEQ ID NO:49)
TTCCTTGNTTTTTANTTGCCAAATNTTACCGATCANTGANCAAAGCAAGCACAGCCA
AAATCGGACCTCACCTTAATTCCGTCTTCACACAAAAATAAAAAAACGGCAAACTCA
CCCCCATTTTTAATTTTGTTTTAATTTACTTACTTATTTTATTTATTTATTTTTTGGC
AAAAAAATCTCAGGAATGGCCCTGGGCCACCTACTATATTAATCATTTTGATAACAT
GAAAAATGATGGGCTCCTCCTAATGAAAAASCAAGGAAAGGAAAAGGCCAGGGGA
ATGAGCTCAAAATTGATGCCCACKTGGGGAGCATCTGGTGAATAATCGCTCACKTCT
TTCTTCCACAGTACCTTGTTTTGATCATTTCCACAGCACATTTCTCCTCCARAAACSC
GAAAAACACAASCGTKTGGGTTCTGCATTTTTAAGGATAARARARARAAAGAGGTTG
GGTATAGTAGGACAGGTTGTCAGAAGAGATGCTGCTATGGTCACGAGGGGCCGGTT
TCACCTGCTATTGTTGTCGCCTCCTTCAGTTCCACTGCCTTTATGTCCCCTCCTCTCTC
TTGTTTTAGCTGTTACACATACAGTAATACCTGAATATCCAACGGTATAGTTCACAA
GGGGGTAATCAATGTTAAATCTAAAATAGAATTTAAAAAAAAAAGATTTTGACATA
AAAGAGCCTTGATTTTAAAAAAAAAAGAGAGAGATGTAATTTAAAAAGTTTATTAT
AAATTAAATTCAGCAAAAATTTGCTACAAAGTATAGAGAAGTATAAAATAAAAGTT
ATYHGTTTCAAAMTAVCDTRTCGAMCTCVTCVABCCCGRGGAAKCCMCTASKKCBA
RHSCGGCCCCCACCSCSSYSKAKMTYCATKCTTTTGAWWCCCTTTAGTGAGGGTTAA NAA-3'
and 5'-CAGCCTCTCACTCTCTNGCT- CTCTTTCTGTCTCTTCCT (SEQ ID NO:50)
CGCTCCCTCTCTTTCTCTCCTCCC- TCTGCCTTCCCAGTGCATAAAGTCTCTGTCGCTCC
CGGAACTTGTTGGCAATGCCTATTTTTCAGCTTTCCCCCGCGTTCTCTAAACTAACTA
TTTAAAGGTCTGCGGTCGCAAATGGTTTGACTAAACGTAGGATGGGACTTAAGTTGA
ACGGCAGATATATTTCACTGATCCTCGCGGTGCAAATAGCTTACCTGGTGCAGGCCG
TGAGAGCAGCAGGCAAGTGCGATGCAGTCTTTAAGGGCTTTTCAGACTGTTTGCTCA
AGCTGGGTGACAGCATGGCCAACTACCCGCAGGGCCTGGACGACAAGACGAACATC
AAGACCGTGTGCACATACTGGGAGGATTTCCACAGCTGCACGGTCACAGCTCTTACG
GATTGCCAGGAAGGGGCGAAAGATATGTGGGATAAACTGAGAAAAGAATCGAAAA
ACCTCAATATCCAAGGCAGCTTATTCGAACTCTGCGGCAGCGGCAACGGGGCGGCG
GGGTCCCTGCTCCCGGCGCTTTCCGTGCTCCTGGTGTCTCTCTCGGCAGCTTTAGCGA
CCTGGCTTTCCTTCTGAGCACGGGGCCGGGTCCCCCCTCCGCTCACCCACCCACACTC
ACTCCATGCTCCCGGAAAATCGAGAGGAAAGAGCCATTCGTTCTCTAAGGACGTTGT
TGATTCTCTGTTGATATTGAAAACACTCATATGGGGATTGTTGGGNAAATCCTGTTTC
TCTC-3'.
[0213] This clone is similar to neuretin (accession # U88958).
[0214] Another EEG nucleic acid clone was designated R233. The
following nucleic acid sequence is within the R233 clone:
46 5'-AAACCNAGAACCCCCCTTTGNAGAACCNTTG (SEQ ID NO:51)
TTTCCTTTCAAGCCCAAGGAAGGCGGGGCCCAACCTTTGGTGTTNTTTGAACAGGCC
TTGAACAGGAGGNTWAGGAGAAATTTCCGGTTGTGGAACCCCAACAGGAACCCCTT
GGCACCCCTGGCCCCAAGGTTGTGMAACTTTGGTTTGCTTAATTTGGACCGTTTTTGC
CTTGAGGATTCATGACTTTTTTTTGKGCCCTTGTGAGCCAAGATGTTGGGTTTTCCCA
TCAACAWTAATAACCCCTTGCTTTTTGGGGTGATTCCCCTGGGGAGTTTCCTGATGA
ATTCCCCCACAGCTCCTGGGGTTTTCATCTTGTTCTTACTGTTGTCTGGATTAGGAGG
GCGGAGAGGGTGGACTCCCTGAGACAAGATAAGCAGGTGGAGACATAGAAGAGGG
AGGGACATTTAACATAGTAACATTTTCAGAGGTGACAGAGATGATACACGGGCAGC
TGGAMTTTTGTGAAGGACAGAGGAGCTGGCAGACCCACAGGGCCATACCTTTGAGG
GACAGGTGAATGGCTGGTTACCAGAGACAGGACTGGTAGACAGTCAAGTACCTCAC
TACGATGTGCCAAGAGATYTGGGATCCTGGGAAATGTGTGGAGAAGAGGATTTGAC
ACTCCCCACCCCCAAGGCCCTTCCCCTTTGCTGACAGCATTGCTGTGGTCGTGGCCTG
TTGCCTTGTCCTCTGTCCCTGGGTGGGGCACACCCTCCTGTGCTGTGCTTGCCTTGTG
CATCAATAAACCAC-3'.
[0215] This clone is similar to KIAA0273 (accession # D87463).
[0216] Another IEG nucleic acid clone was designated R241. The
first library screen produced a clone designated R241-4. This
R241-4 clone contained a 2.0 kb fragment and a polyA tail. A second
library screen using 5'-end of R241-4 as a probe produced an
additional clone designated R241-12. The following nucleic acid
sequence is within the R241 clone:
47 5'-GCANTTTGGAGT (SEQ ID NO:52)
TATTGCTTAAAACCAGGNTAAGGCACTTTGTCCCACAGGACCCAGGAATCNTAAAN
GGGTTGAAATTGGGNCGGGGAACCCCAGGATATAATGCNACTTTTGTTAGGGGGAG
AGTTCAGCTCTAACTGGTAGTAGTGTGAAAGTAAGCACCTTGACTTCAATTTTGGAA
AGCACTTGGTAAATGGAGAGAACTTTGGAGTTTCCCTATCATCTATATCAGTCTTTG
AACACACCCTCAAGTCCCAGCCTCAAGGCTCAATAAAGGACCACATAGCAGGTCTG
AGGCTCACTGCTCTCAGCCCTTAACACAGGGCAGTGGAGAGCAGGGTGATCTTCCCT
CTCTGGAGCTTCTCCTTGGCCTTCTTCTCCACTTGGGCTTCTGCTCAGCAGCAGATAT
ATTCTGGGTTCCATAAGGAATCCAGCTGTCCCAGTGGCTTGACCCTGTCAAGGCAAG
ATATCAACTCTGAGGATGACCCAGTCATGGAGGAAGAGAGTGTGACAAGATCCGCA
GTTTGAAGCAAAACTGTGTTTGGTCTTTTCAAGAAACAAATGGGCACATTGAGTTCT
GTTCAGTGTCAGAGGATATCTTTCCCTTTGCTCCCAGATTTCCAGAAATGGATAATGT
TTTCATTTCTGTGGGAAGGGTCAAGAAACATAAAATTGCTCAACAATGCTTGCTTCC
CTTGAGGGTTGTTGAGCAAAGGCCGATATGCCTCCCTGCATTCTCTTCTACCTCAAG
ATTTTGGAATTCAATTCTGGAACAGAAATTTATTTACACAAGAACACTTGTTGTCAG
CCTTGGTTACTGTGGGAGTTACATAAGGGTGACAGTCTGTATCTTCTAARTTAAACA
GGAACTGGGCTTTGGCGGCCTATTGACCCAGTTTATATCTAAATATAACTGTGGCTC
CAAATGATTGGCCAATAACATTCCCTTTACCTTCAAAGTTTTCTCCATCAGTCATTTC
TGTGGCAGCACAGTTCCAATGTCATATGCCCC: TGCAAAGTTGAAAGTAATTAGTGA
CAAAATAACCCTCCCCCCTTTCAGTGGCCAAACTGTCAGCTGTAGCAGCGCTGCGAA
AGCGAGTACTACACTATGTACGGAAAG: CCTGTTCCTTATCACGGACTAGACTCAAG
AAATGCCATCTCCGAACGGTGGCATTCAAGGTGGTAGTCGTTTGAATGGAACAGTCA
TCTATGTGGACATTGTTAAAGTGTTTTAAAGAGTATTTTGAAAATTAAGTTTACATTT
TACAACTGCTTTATTTTTATTGAAACAATTGTATATAAATATTACCCTCTTTCACTGTT
AATTAAAGTAAACCTAGACCTTGTAGACAAGTGGGTCAACTGATATGTATAGAAGCT
GTGATGTAGACAATACCTTTCTCTTGTGTAAATGGTCATAAATATAGCTGTTCCTGTG
TTTTTATAAGTTGAGGGTATTTTGTTGTTTTATAACAACAAAATTTATTGCATTTGAA
ATGGTTTTTATGTAATAGAATCATGCAAACAGTGAAGGATTATAACATGGTATATGT
AAATGTATAAACTTTAGAAAGAAATAAATACAACAAATTTCAAAAAAAAAAAAAAA
AAA-3'.
[0217] Northern blot analysis using the 3'-end the R241 clone as a
probe revealed the presence of two mRNA transcripts: one about
7.0-8.0 kb and the other 4.8 kb. In addition, this analysis
revealed that the expression of the R241 mRNA was marginally
upregulated in response to the multiple MECS treatment.
[0218] Another IEG nucleic acid clone was designated R256. The
first library screen produced a clone designated R256-8. This
R256-8 clone contained a 1.8 kb fragment. A second library screen
using 5'-end of R256-8 as a probe produced two additional clone
designated R256-2 and R256-3. These additional clones contained
each contained a 3.0 kb fragment. The following nucleic acid
sequence is within the R256 clone:
48 5'-GGCACGAGGACAGATTCTGAGATGGAAACTTAAATTACATCCCAGAGGCAGG (SEQ ID
NO:53) GAAACTATGAAGTCACCGTTCCTAGACCACCCCTTACTGAGGTTCCACG- GTCACACTG
ACGGCAGGACCCACAAGGGCAGGGTATTGGTCTGCCCTCCTTTCTCCT- GTCTGTCTG
ACTTACCTAACTTTGGTCTCGGCTGCTGACACTTGGAAAGGACCAAAT- TACTTGATAG
TATTTCCCCCTGTTTGTGTAATAGCCTGAAACCTTGGAGAGGTTCCA- GAATACTTCT
GTATATAGGGCACAGGTGAAGACATTGTCCAAAGCTTATTTATTTAT- CTATTTATTT
ACCCTGGCTGAGTAACCACACCAGTAGGGGGAAAACTAAAATGTGTT- GAGTGTAAA
CAAAGTCACCAGCCTGGCTAGAAATTCTCCCTGGAAAACATCCATTTT- GATACAA
TGTAAACGTTAGTGTTCACCCTTAGATACATGTTGAAAGAGAGCTTTGGT- ACGCG
GAAGTGGCATCTTTGGTCACACACCATGCCAAAGTGAAGAGGTGGCCAGTGG- AGGTC
TTCCGGTCCTGTCGGGATCATTTGTGAATACATTCTTTGCCCCTCTTAAGTA- CTTGTT
TACTAAACATGTGCAGTGGTAGGTATTAGTGTTAGATCACAGTGGGCACTT- CCCTGGG
GATCTGGGGAAGACCAGAGCTTGCAACTCTGCCTGTTTTGATCCCTATTT- CTCACAG
TGCTGTATTAAAAAAAATAGGATTTAAGACAGATAACCACCTTTACATTG- TGAGTGT
GTTTGCCTTGTCTAACGACAGATAATTCTTAACATTTCTCTTCACCTTAG- TACTTT
AGGCTAATTATACACGTCTGTCTATGCCATGAGTAAGTGGACTGTAGTCGG- ACCAAA
AGAAAACAAATGAGCCGTTGGACCATTTGTGCAGTCAGTTTCTGGTCCTTA- GATGT
ATCCTAAGCAGTAAGTGTCTGATTGTACCCTGGTGGTATGATCAGTTGTCTC- GTAG
CTGTCTCAGCTCCACAGTTTACAATGCAAATCTGTCTCAAGATCTTCACGTCA- CTG
CTGCTGAGAGCAGGGAGAATTCTCTGCAGCTGTTTCAAAGTTGTGGCCCGGCCT- TG
AATCCTCTGTTAATTACTGTGTGAGCCAGAGGGAGCTGCCCAGCAAGGGTGGGCC- C
CCAGCCGGCAGGGGAACTTTCTAGACTCCCCGCTCATTCAATTGATCTAGGCATT
CGGGCCTGCTACTTGACCATTCTCGCCCTGTGAAATGTCCCACACTTTGAAGCAAA
TACAATTCACAGCACAGTACACACAAAAACCCTGGCATAAGACAGGGGAGGTTC
TTCTTATTTTGTGAGCCGGTTGCCCTGGAAACGGATAACAAAGGGCAGCCTTCC
ACTTCTGGCATAATGGTGGAGCCTCTTTTCTCAGGCTTGACACCTGTCTGAATA
AGAGTGATTAGAGCCGCATAATATCCCTCTCTTGGCTATTGAATATGTGGTTCACA
TACCAAACCCTGTAGAAGTTAGAAGACGGTCGTGTTCGTATGTTGTTTGCTTCCAC
TACATTTTTGAGGTTTTGTAAAACTGTTATTTTTTTTCACGATGTGAAACTGAA
GGTCAATAAATTATTAGAGATTTTCAAAAAAAAAAAAAAAAAAAAA-3'.
[0219] Northern blot analysis using a sequence from the R256 clone
as a probe revealed the presence of a 4.0-4.8 kb mRNA transcript.
In addition, this analysis revealed that the expression of the R256
mRNA was moderately upregulated in response to the multiple MECS
treatment.
[0220] Another IEG nucleic acid clone was designated R261. The
first library screen produced a clone containing a 1.0 kb fragment
with a polyA signal and tail. A second library screen using a
portion of this clone as a probe produced 41 positive signals that
were isolated. In addition, PCR using T3 or T7 primers along with a
R261 sequence specific primer resulted in the 850 bp of additional
sequence from a solution containing the phage plug from a first
screen. The following nucleic acid sequence is within the R261
clone:
49 5'-CTTAAAACCCCTAGATTTCCTGTTACATACTAACACAGGTCTTCCCTTTCACT (SEQ ID
NO:54) CCAACCCCAGGTTTCAGGCCTCAGAGCCATGCTGGGGTTGGAGAAAACT- GCATTCCTA
TGAGGGTAAAAAGTAGCTGCCCTCTCTGACCCTTTCTTGCTAGGCTTC- ATGCGGGAT
GGGAGAGGGTATCCCCAGGATGGGGACAGAGGAAGCCTGGCTAGGGCC- TTCTAGCC
CAATAAGCCAAACAGGAACTATAAGCAGATCAAAATCCTACACTAGCTT- ATTAGGGC
CCTGTTAGTTGAAAACCTTGTTGCTGTCCCAAGTTCTTCAGTTACAACC- GAGTACACT
TACTCTTCCAACTGTCCTAAGGGTCACTACCCAGCCAGCTTTGGATCT- TCAGCACTTT
TAAAAGCTGAAACTCCCTCTTGCCCTTCTTGTCTATTCCTCACTGCC- AGTTGGGGCCTA
GGCTCAGTCCTGGGCAAATGCCCATGATCCTGCTGCTGTGGGAAG- TTTGATAGGGCAT
TTGGCTCAAATTTCAAAAGGCCTCGCTCCTGACCTGATTTCTCG- AAGCTCCAGTAGTT
CTAGACCCCTCCAATCTCTCATCTGACTGGTTGCAAGGCTTAT- TTTTCTTTTGTACTT
TCCTATAGAGCATTTCTGTAGCATTTGAGTGTGGCGATATTT- TTGTTGTGTGTAG
ATTTCTAAGAACCAACACTACTCAGTCTCCTGCTAGTCTGACTC- CTGAAGCATCAGAC
CTCGTCATACGGTATTGACTGTGTATGTGCCTTTCACCTTGAG- CATGCTTCAGGATTT
TTTTTCTTAAACCACAGAACTTGAATACACAAGGGAACCAGA- ATTCACAAAGTCCTAT
GCAACCCTAGACAGGAGGAGGTTAGAGAGTCTGTCTTGATT- GGTGATTTCAGAGAC
CCNAGAGAAATTTGTACCAGTTTGTATTAATGTCAGTACTAC- CAGCACTTTGCCAAAA
CTAAGGATGTCAGAGGGACCTGTTTCTAGAGTGAGTCCCAA- TTACATCAAAGGGCAA
CTTACAGCTTTCTCCAGTAAGTCTGAGTGGTTCTCTTGAGC- TGGTGTCACTTTC
TAACCTTTGCCAGTCTAGCCCAGCAGGGCCCTGTGTGTGTGAGT- GCAGTTTGGTGCT
GTTTTGGAGTATGCCTGCTCCCCAGCCTGGAACCCTCTCAGCAA- CTTGCTGGGACCT
ATAATGTCTTAGGTGCAACAAGGACCCTACCAGAGCTCCTGGGT- GGCTTTCAAGATC
CACGTAGCTTTGTGTGAGGGGACTGAATGCAGACAAACCACAGC- CTGCTTCAAATAC
CTTCTTTCCCTACCACCTAGTTCCAAATGGAACCAACAAGTTGA- GTGCATCTCTGTT
GGGTGTTTTGTGTTGAGACTGGCTGAAGTGAAAACTCTTTGACT- GACCATGTTGTGAT
GTGTCGACAGACTCAAGGACACAACCACCTCGAGCTGGTCATG- TGGCATGCCTGTGT
ATGTGTGTAACAGGATTCTGAATGTTAGGTTGTAATGCTATTC- CTGTATGGGAGAAAA
AAATAATATAAACAAATAAAAATCTATTTAAAGCACAAAAAA- AAAA-3'.
[0221] Sequence analysis revealed the presence of some homology
with EST sequences including that of a cDNA clone from ae69b04.s1
Stratagene schizo brain S11 (accession # AA774320).
[0222] Northern blot analysis using a sequence from the R261 clone
as a probe revealed the presence of a 4.0 kb mRNA transcript. In
addition, this analysis revealed that the expression of the R261
mRNA was marginally upregulated in response to the multiple MECS
treatment.
[0223] Another IEG nucleic acid clone was designated R272. The
first library screen produced a clone that was used in a second
library screen. This second library screen produced two additional
clones designated R272-1 and R272-2. Clone R272-1 contained a 2.0
kb fragment while clone R272-2 contained a 1.7 kb fragment. The
following two nucleic acid sequences are within the R272 clone:
50 5'-CCATGGGGACTGGTTTGTCACCNATTGCCCATGGNTTGGTTGGTAG (SEQ ID NO:55)
GTGTTTTTTGGTGGACATTTTTGTTTCNCGTTTTGAACTCCAGATTATTGGGTTTT- TG
TTTTAATTTATTTTTGTCAGAGGAAAAATAATTTAACATCCATCTCACAGGCTTG- CTT
GACTGTTCAGTTCCAAGGTCCTGCTCACTTTTTCTTGTCTTGCCTCTGCTCTGG- CTTT
CTTCATGATAGTGCTGGACGTGGAGCTGAGAGTCTCGTTTACTCTAGGCAAAC- CCTCT
ACCTGAAGCCAGAGCCCAGCACTCCGTACCACCACAGACTTCTGAAGCTGGC- AAAGTT
TTAGAAGCTGGGAGTTTTCTGATTCTCTCATTATTAAGTTTCTCCTCAGTC- TTTAGATA
GAGGTAAATGTGGGCTTGTAAGAAAAGAAACGAAAGCACGTAATGTACA- CCTATTCT
GAATTATGCAAATTAGCTCTTACTCAGGGTCAACTAAATTACTTCAACT- CGCCCTTTA
GTTTACTCTTAATTTGCAAAAAGAGAAAAAAGAAGGAAAACTAAATAG- GACTATGAT
TTGGGGAGCCAAATTGATAATCTGATGTAAAAGTTGCTGTGTTAAACA- TAAATTATT
AAGTGTAGACTTTTTTCCTAGGATATTGTATTCATTTTGTGATATCGC- CTAGAATGAT
GTATTAGATAAAAATCAATTTTGTAAGTATGTAAATATGTCATAAAT- AAATACTTTGA
CTTATTTCTCAAAAAAAAAAAAAAAAAAAAA-3' and
5'-GATTTTATATTCAATGTTGTTTATTTAATCCATTGCAGTTGGTGAATGCCTTTT (SEQ ID
NO:56) CCTCCTAGACACCCTGTATTATACCATTTGGGGATTAAGTCA- AAGTTAAGTATATTTT
TTTCTTACTTGAGCTCTATATATGCAATTCAGATATCTTCC- TGATGACAGTTTTATAT
GTAAATGTAATTTAACTTTCTTTCCGTGTTGACGAAGTTC- TGTAGGTGTTAGGGTTAG
AAGTCTCAGCACTCACTTCTCTCACTGGATGTGCAGTGT- GCCTGCCATGGCGCACGG
CTTCTCAGTAATGATGCCATCTCTGCTACTTTTACAGAA- GGAGAAGTTTACTTTTGAG
GTGGGTATGTGTTGATATCTAAACACTGTGTGTTGCTT- GCTTAGATAGGCAAGACAC
ACTGCTGTGCGTGGCTCCTGTGGTGCACCTAGCCCAGG- GGAACGTAGCCTCAGTACT
TCCGCTGGCTTCTTCATGCCTAAGAAGCAGGGGCCTTT- CTTGTTTGCTGGGCTCTGGC
TTTAAAAGTTGTCCTTTGGGTCTGGAGATGTAGCTCT- GTGACAGAACACCAGCTAAT
GTCAGGTCCTGCGGTCAGTCTCTGGTACACACAAGCG- CACACTCACATGATGGGGGG
ATGAAAGGCTGTCCTTGTGTAACAGTATTCGATGGGG- CGTTGCCTGGATGACGATGT
TTATGTACTCTGAAGGCAGATCCTGAAGGCACCCTGT- TCTTCCCTTCCTTGTGTAACT
GAGTCTGCACTAGCTTAGCCACTGTTTTAGAGGCCA- TCCTAGTGGGCGAACAGGAGG
CATCGCACTGGGTGATGGTTTGCCTTCAGTCCTCAA- GTAACAGCGGCCGACTTATGC
CGATGGCTTGTTTGAAATCAAATATTACCAAGTTGG- CCTAGTCTGCCTTCTGTGAAG
AAGGGGAGAAAGGAAGGGTGGAAAGGTGGATGGAAA- GCCTTTGGGGAACTAGTCT
GATCTCTCAAGGG-3'.
[0224] Northern blot analysis using a sequence from the R272 clone
as a probe revealed the presence of a 1.0 kb mRNA transcript. There
appears to be a discrepancy in the length of the R272 mRNA since
the Northern blot data indicates a message of 1.0 kb while the
cloning data reveals a message length around 2.0 kb. Regardless,
the Norther blot data indicated that the R272 mRNA expression level
was moderately upregulated in response to the multiple MECS
treatment.
[0225] Another IEG nucleic acid clone was designated R280. The
following nucleic acid sequence is within the R280 clone:
51 5'-CTTCAGTTCCTTTGAGGGGNCTTTCCTTCGAAGGGGATACGCCTACCTTTCACGA (SEQ
ID NO:57) GTTGCGCAGTTTGTCTGCAAGACTCTATGAGAAGCAGATAAGCGAT-
AAGTTTGCTCAA CATCTTCTCGGGCATAAGTCGGACACCATGGCATCACAGTATCGT-
GATGACAGAGGCA GGGAGTGGGACAAAATTGAAATCAAATAATGATTTTATTTTGAC-
TGATAGTGACCTGT TCGTTGCAACAAATTGATAAGCAATGCTTTTTTATAATGCCAA-
CTTAGTATAAAAAAG CTGAACGAGAAACGTAAAATGATATAAATATCAATATATTAA-
ATTAGATTTTGCATAA AAAACAGACTACATAATACTGTAAAACACAACATATGCAGT-
CACTATGAATCAACTAC TTAGATGGTATTAGTGACCTGTAACAGAGCATTAGCGCAA-
GGTGATTTTTGTCTTCT TGCGCTAATTTTTTGTCATCAAACCTGTCGCACTCCAGAG-
AAGCACAAAGCCTCGC AATCCAGTGCAAAGCTTGCATGCCTGCAGGTCGACTCATAT-
GCGGTGTGAAATACCG CACAGATGCGTAAGGAGAAAATACCGCATCAGGCGGCCATC-
GCCCTGATAGACGGTT TTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGG-
ACTCTTGTTCCAAACT GGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTT-
ATAAGGGATTTTGCC GATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAA-
ATTTAACGCGAATT TTAACAAAATATTAACGCTTACAATTTGCCATTCGCCATTCAG-
GCTGCGCAACTGTT GGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCT-
GGCGAAAGGGGG ATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCA-
GTCACGACGTT GTAAAACGACGGCCAGTGAATTGTAATACGACTCACTATAGGGCGA-
ATTGGGTACCG GGCCCCCCCTCGAGGTCGACGGTATCGATAAGCTTGATATCGAATT-
CGGCACGAG CCGCAGCCGATATGCAGTCCCCGGCGGTGCTCGTCACCTCCAGGCAAG- TTCAGAA
TGCGCACACGGGYCTCGACCTGACTGTACCACAGCACCAGGAGGTGCGGG- GT
AAGATGATGTCAGGCCATGTGGAGTACCAGATCCTGGTGGTGACCCGGTTGGCTG- TG
TTCAAGTCAGCCAAGCACCGGCCCGAGGATGTCGTCCAGTTCTTGGTCTCCAAAA
AATACAGCGAGATCGAGGAGTTTTACCAGAAACTGTACAGTCGTTACCCAGAAGC
CAGCCTGCCCCCACTGCCTAGGAAGGTCCTGTTTGTCGGGGAGTCTGACATCCGGG
AAAGGAGAGCCATGTTTGATGAGATTCTACGCTGTGTCTCCAAGGATGCCCAGTTGGC
GGGCAGCCCAGAGCTGCTAGAATTCTTAGGCACCAGGTCCCCGGGGGCTACAGG
CTTTGCCACCCGAGATCCCTCTGTCTTGGGATGACGACAGCCAGCCGGCCAGGGGAC
AGTGATGAGGCTTTTGACTTCTTTGAGCAACAGGGATGAAGTGCAAGCCACCCACA
TTGGGCCTGAGCAACAANGAAATGTTGAGAAGGTCCNTGGAAGGAANGAGGAGG
GAAGGGAGGAAGGANGATAACTTGGGATCCCCCTTGGGGCAATCAATGCGGCCT
CCCAAAGGAAAGNCCCTAAAG-3'.
[0226] Northern blot analysis using a sequence from the R280 clone
revealed that the expression of the R280 mRNA was upregulated in
response to the multiple MECS treatment.
[0227] Another IEG nucleic acid clone was designated R286. The
first library screen produced a clone that was used as a probe for
a second library screening. Briefly, the .sup.32P-labeled probe was
used to screen a UniZAP rat hippocampal oligo(dT) primed library
(Stratagene). This second screening produced a clone having a 4.7
kb full-length R286 cDNA sequence. The nucleic acid sequence of
this rat version of R286 is as follows:
52 5'-CTGCCAGCCGAGGCTCCTGCCGCTGTGACCCGCGCTCCGCCCGCCGC (SEQ ID
NO:58) CGGGCCGGGACCCTGATAGCTAATGTCAGAAGAAAGTGACTCTGTGAGA- ACCAGCCCC
TCTGTGGCCTCACTCTCCGAAAATGAGCTGCCACCGCCTCCCCCGGAA- CCTCCC
RGCTACGTGTGCTCGCTGACAGAAGACTTGGTCACCAAGGCCAGGGAAGAG- CTTCAG
GAGAAGCCCGAGTGGAGACTCCGGGATGTGCAGGCCCTTCGAGACATGGTA- CGGAA
GGAGTACCCATACCTGAGTACATCGCTGGATGATGCCTTCCTGTTGCGCTTT- CT
GAGGGCCCGAAAGTTTGATTATGACCGGGCCCTGCAGCTGCTGGTCAACTACCAT
GGCTGCAGGCGGAGCTGGCCAGAGGTCTTCAGCAACCTGAGGCCATCAGCCCTGAAA- G
ACGTTCTTAACTCTGGATTCCTCACAGTGCTGCCCCACACAGACCCCAGGGGCTG
CCATGTCCTCTGCATCCGACCAGACAGATGGATACCGAGCAACTACCCGATCACC
GAGAACATCCGCGCCATCTACTTGACGTTAGAAAAACTCATTCAGTCCGAGGAGAC
CCAGGTGAACGGGGTTGTAATCCTCGCCGACTACAAGGGAGTGAGCTTATC
AAAGGCGTCTCACTTTGGCCCCTTTATCGCCAGAAAGGTGATTGGCATCCTT
CAGGATGGCCTTCCCCATTCGGATAAAAGCAGTTCACATAGTAAACGAACCTC
GGATATTTAAGGGCATTTTCGCCATCATAAAACCATTTCTGAAGGAGAAAATTGCAA
ACAGGTTCTTCCTCCATGGGTCTGACCTGAGCTCTCTGCACACGAGCCTTCCAAGGA
ATATCCTCCCCAAAGAGTATGGGGGCACCGCTGGGGAGCTGGACACTGCCAGCTGG
AACGCGGTGCTGCTGGCCTCGGAGGATGATTTTGTGAAAGAGTTCTGCCAGCCTGAG
TCTGGCTGCGATGGTCTCTTGGGCCAGCCCCTGCTGCCTGAGGGGCTGATCTCAGAC
GCGCAGTGTGACGACTCCATGCGAGCCATGAAGTCCCAGCTCTACTCCTGCTATTAG
CCCTCTTCCGGGAGAATCACCATGTGTAATTCCTTCCTTCTTCGAATGCACAGGCTGA
AGATGCCAGGACCTCGGTCTTGCTCCATCACAGTGCAGCACGGAGCTGCCTGCAGAG
ATTTAAGGAGAGCCCATCACAGGCAGACCTCTGACCAGCTAGGTTATTCCAAGAAG
ACATGGAAATTGCCCTGGTGATTCCCAGATGTCTGTACTCTAAGTCTGCAACTGTTA
CTCTGGAAGCTGCATCTGTTTCTTATGCATCTTGGAAAGAACTAGGGTCAAAGTCAC
TCTGAAGTGACCAGGAGTAGACAACTTGATTGATCATGAGTCTGAAACAATTGCCAA
TCCTGAAAGGTGGCCATGCGTGAGACTTTGAGTCTCTTTCCCATAAACTGTAGGTGT
TGACTACTGCTGCTTATCTGCAAAGGTCAGGGTTCAGGCCCCAGTTGGCATTGCTGG
GTCTGGGAAGCACTGCTAACTGAGTGGTAGAAACGCCAGGCCCAGGCAGCACTTAA
AGGTTAAAGGTCAAATTTGGAAGCTAAGGCTATAAATCATCCTGGGTTCCAGGCTTA
AATCTTGCAATGGACACTCTCCCCAAACCATAAAGCCTTAGCTCTGGTTCTCCATGG
AATCATGCAGGTCAACATAAAATACTGGATTCTTGGACTGCGTGGCTAAAAGCACTT
AGACTARGAGTCCAGTGTGTGACTGGATGGATAGGGGCCTCAGCTTGTCAACTCTAA
GTTAGMGMTCCATGGAATGAAGGCCTTGRGGGCTGCTCAAGTTCTGTTAGGTTTCTG
CTTGGAAAGATGACCACCTGGAGGTGGCCGGGCCTTTTTGGTTTGGCTTGGTTTTGT
GTTATAGACACAAGCCTTATGGAAAGGAACCGTCTGGCCTTTAAAGAAATTACTATG
TTCCTGGGAGTTGGTGGTAACCAGCTGCTTTTGCAGATGATGGGTGAACTGGAAAGG
GATGGCTTTTGTGAGGCTGACCAAGTCTTGTACGCGGATGTTGTACAGATTCCTCCC
ACACCGGAGACATTCGTACTATATTAGAAACAGCCACGGACTTGTGCTCTTTCAGTT
TGTGTCCCTGGAAACATACGGGGGGCAGGCTGTTGCTGGTTCACCTGGGGGCCCTGC
CCTCCCAGACACGGGAGTGCTTGTCTAGCGTGGGAGGGCCAGTTGGCCAGATTGTTA
GCTCTGCGTTGGGGTGTCGTAGACAACTGACAGGATTTTAGCCTTAACCCAAGCACT
GAGTGAGGTGATTTTTCCCTTGGCTTTTGGCGTGTCTTTGGTATTCACCATGTATTGT
GGTGTCAGGTAGTGTCAGGTACTGTTGGCTGTGTGTCTCCTAGACTAAGCGGGCGTT
GSATACAGCTTACATACAGTGCTTGGAGACCAAAGGTCAGTTGGTTGTAATAAGCTG
GTCCACCCTTAACAGACTTCCCAAACATYACAGAAGCTYTTATGGMCCTTACCTAAT
AATGCCAATTCTGGAGGACACTCTTTTACCATAGAWKCSAATCCTTGATCTCCTGGC
TCCTGGTTGAGCTTCCGCACTGATACACCCTCTTGRCTGCCCATCAGGGCCATTTGCT
GCTGAGTTCTGCATTGCTTAAKCTSCKGSYGYTTTCTGCCTAAAGGGATGGCCACCC
AGACACCTAAAAAGACCCGGGATGGCTCTCTAGCCTTGGTGGAGAGTCTTATTAGAA
GTTTTCTTTGGGGGATTGGGGATTTGGCTCAGTGGTAGAGCGCTTGCCTGGCAAGCA
CAAGGCCCTGGGTTCGGTCCCCAGCTCTTAAAAAAAAAAAAAGTTTTCTTTGGTAGT
TGGGGAAAAGGCAGAAGGAAAAAAACAAAGGGAAAGATGAATCTCTCAGTCCTAC
CTGGTTCCCTAAATTTAAATCGTGTCATGTGACTAGTTAAGTCTCTTTGACTTAACAA
AGGGACACCAGGTTCTTGGGGAGAAATCTCAGAGCAAAATGTTGCCTGTTGSTAACC
TTCTGGTAACCARAGGARCCTTGATAARCTTARGAGYKGACTGTATGTCCATGCTCT
TGTGACTCTAGAGACTCTGGCACCTCAGGTTNAAGCAGGCTGTGAGCCAGATGTCCT
GGTGCCAAGCAACCCCACTGTTGAGCAGCAGGGGCACCATAGGCCTCAGCTAGGGG
AGCGCACTGGTAGAGCCAGCAAGTGAGCAGGAATCTGACTTTAGGGTAAAAATCTA
GACAGTTCTGACAGCTGGAAGTCAACTTTTCCTCCATTCAAAGTCATGTGGCATTGG
GAAGGGGCTAGGGAAATAGAAGTGGGTTCCAGCTTTATCTTCCTACACAGTCTCGAG
TATAGCATTAACACCGAGTGCTGGACAGAGGTTGTCTGCTGAACACTCAATCCTGCT
CCTGACTGACTCTGGAAATAAGGACATTCCACTCTGCTTGGCGCGGAGATGCCCTAG
TGTGCGGCCGCGGGGGCTTCTCTTTCTCAAGTCCTCTACAGNACTTCCAGGCAGTTC
ATCTTCCTAGGAAAAGGTATGGAGGTTCTGCCTTCATGGTAGAAACACAGGATAAA
ATCTACAGTAAACAACCGGTAAGTGCTGGCTTCTTACGCCTTGGCTTTCTCCAGGC
CAGGTGGGTTCGACTACTCCCATTTCATCTTTGTAAGCACCTCAGGTTATAGGGCAG
TTTCTTCAGAGTTGGGGGGACTGGAGCCATTCCCCCTGTAATGCCTGAGGTGGCCTT
ACCACCTAGCAGCCAGTTTGGCCAGCAACAGCCACACTGCTGTTATGGTATCATAAT
ACCTCATCCTCGGGTTTCCTTCAGAAAGGRAAAWGCTAACTCAGTTGATGTAAGTG
TGCTGTGCTGGGATCCTGTCATGTGGGAGGGAACACCAAATACACAGGCTCTCAGG
AGACATCTTGCTAAGGCTTCTCTTTACTGCAGTCTGCTCACGTTGTAAATCTGCCCTC
TGTTCTCCTGACTCARAAAGACTCAGCCMCAAATCAAGAAGCGCCATCAAACGTTCC
TTCTCAKKGGGAACGTGCTCCACAGGAAGGTCCAGWGGGATTTGCARCTAGAGTCA
CGTTTTACTGGKTTGTGAMCAAATTTACTGGTTTTCARTTACCTGGGGKCCTATGKG
KKTTTTMAACCTTTTCCCATMAGGCAGTTAGTAGTAGCCACTTTGGGTTCCTGTGGA
CGTGCCTCAGCTTCTCGGCATAGGAACCCAACAGGTAGAATACTTGAAACTTCTCAG
TGGCCAAGACCTCGATACCCTCTCTGATGGGTGGGAACTGGGCTATTTTCCTGACCA
ATCTAGGCCACCATTTTAGTCCCTGGTCACATTCCTTACTCCAAACTGAAATTCAGTT
TGGCTTTGAGTATGTGCACACGTGGTGGGTTCACCTACTTCAGTGTTGACCAAAAGT
TTATTTTTCTAGTGCATTTTTCTAAATGGTAAAAATATGTAATTTTAGTATGCATGAC
TGGGTCTCCAAAATAAAAACTGAGTGTATTGTGAAAAAAAAAAAAAAAAAAAAAAA
AAAA-3'.
[0228] The following nucleic acid sequence is the ORF for rat
R286:
53 5'-ATGTCAGAAGAAAGTGACTCTGTGAGAACCAGCCCCTCTGTGGCCTCACTCTC (SEQ ID
NO:59) CGAAAATGAGCTGCCACCGCCTCCCCCGGAACCTCCCGGCTACGTGTGC- TCGCTGAC
AGAAGACTTGGTCACCAAGGCCAGGGAAGAGCTTCAGGAGAAGCCCGAG- TGGAGAC
TCCGGGATGTGCAGGCCCTTCGAGACATGGTACGGAAGGAGTACCCATAC- CTGAGT
ACATCGCTGGATGATGCCTTCCTGTTGCGCTTTCTGAGGGCCCGAAAGTTG- ATTATG
ACCGGGCCCTGCAGCTGCTGGTCAACTACCATGGCTGCAGGCGGAGCTGGC- CAGAG
GTCTTCAGCAACCTGAGGCCATCAGCCCTGAAAGACGTTCTTAACTCTGGAT- TCCTC
ACAGTGCTGCCCCACACAGACCCCAGGGGCTGCCATGTCCTCTGCATCCGAC- CAGAC
AGATGGATACCGAGCAACTACCCGATCACCGAGAACATCCGCGCCATCTACT- TGAC
GTTAGAAAAACTCATTCAGTCCGAGGAGACCCAGGTGAACGGGGTTGTAATCC- TCG
CCGACTACAAGGGAGTGAGCTTATCAAAGGCGTCTCACTTTGGCCCCTTTATCG- CCA
GAAAGGTGATTGGCATCCTTCAGGATGGCTTCCCCATTCGGATAAAAGCAGTTC- ACA
TAGTAAACGAACCTCGGATATTTAAGGGCATTTTCGCCATCATAAAACCATTTC- TGA
AGGAGAAAATTGCAAACAGGTTCTTCCTCCATGGGTCTGACCTGAGCTCTCTGC- ACA
CGAGCCTTCCAAGGAATATCCTCCCCAAAGAGTATGGGGGCACCGCTGGGGAGC- TG
GACACTGCCAGCTGGAACGCGGTGCTGCTGGCCTCGGAGGATGATTTTGTGAAAG- A
GTTCTGCCAGCCTGAGTCTGGCTGCGATGGTCTCTTGGGCCAGCCCCTGCTGCCTG- A
GGGGCTGATCTCAGACGCGCAGTGTGACGACTCCATGCGAGCCATGAAGTCCCAGC
TCTACTCCTGCTATTAG-3'.
[0229] Using the rat R286 cDNA sequence and a portion of the human
R286 nucleic acid sequence, specific primers were designed to
amplify the human R286 homologue. After RT-PCR using human
hippocampal RNA and the specific primers, the PCR product was
subcloned in the TA-cloning vector (InVitrogen) and sequenced with
SP6 and T7 primers. The following nucleic acid sequence is the ORF
for human R286:
54 5'-ATGTCCGAAGAAAGGGACTCTCTGAGAACCAGCCCTTCTGTGGCCTCACTCTCTGA (SEQ
ID NO:60) AAATGAGCTGCCACCACCACCTGAGCCTCCGGGCTATGTGTGCTCA-
CTGACAGAAGAC CTGGTCACCAAAGCCCGGGAAGAGCTGCAGGAAAAGCCGGAATGG-
AGACTTCGAGA TGTGCAGGCCCTTCGTGACATGGTGCGGAAGGAGTACCCCAACCTG-
AGCACATCCCT CGACGATGCCTTCCTGCTGCGCTTCCTCCGAGCCCGCAAGTTTGAT-
TACGACCGGGC CCTGCAGCTCCTCGTCAACTACCACAGCTGTAGAAGAAGCTGGCCC-
GAAGTCTTCAA TAACTTGAAGCCATCAGCCTTAAAAGATGTCCTTGCTTCCGGGTTC-
CTCACCGTGCTG CCCCACACTGACCCCAGGGGCTGCCATGTCGTCTGCATCCGCCCA-
GACAGATGGATA CCAAGCAACTATCCAATTACTGAAAACATCCGAGCCATATACTTG-
ACCTTAGAAAAA CTCATTCAGTCTGAAGAAACCCAGGTGAATGGAATTGTAATTCTT-
GCAGACTACAAA GGAGTGAGTTTATCAAAAGCATCTCACTTTGGCCCTTTTATAGCC-
AAAAAGGTGATT GGCATCCTCCAGGATGGTTTCCCCATTCGGATAAAAGCAGTCCAT-
GTGGTGAATGAA CCTCGAATATTTAAAGGCATTTTTGCCATCATAAAACCATTTCTA-
AAGGAGAAAATA GCAAACAGATTCTTCCTCCATGGGTCTGACTTGAACTCTCTCCAC-
ACAAACCTTCCA AGAAGCATCCTCCCCAAGGAGTATGGGGGCACGGCTGGGGAGCTG-
GACACTGCCAC CTGGAACGCAGTACTGCTGGCTTCAGAAGACGATTTTGTGAAAGAG-
TTCTGCCAACC TGTTCCTGCCTGTGACAGCATCCTGGGCCAGACGCTGCTGCCCGAG-
GGCCTGACCTC AGATGCACAGTGTGACGACTCCTTGCGAGCTGTGAAGTCACAGCTG-
TACTCCTGCTA CTAG-3'.
[0230] The R286 clones were found to be homologous to a family of
transfer proteins for hydrophobic ligands (such as lipid soluble
vitamins and phospholipids). Thus, R286 is a lipid transfer
polypeptide. The amino acid sequence of the rat R286 polypeptide is
as follows:
55 MSEESDSVRTSPSVASLSENELPPPPPEPPXYVCSLTEDLVTKAREELQEKPEW (SEQ ID
NO:61) RLRDVQALRDMVRKEYPYLSTSLDDAFLLRFLRARKFDYDRALQLLVNY- HGCRRSWPE
VFSNLRPSALKDVLNSGFLTVLPHTDPRGCHVLCIRPDRWIPSNYPIT- ENIRAIY
LTLEKLIQSEETQVNGVVILADYKGVSLSKASHFGPFIARKVIGILQDGF- PIRIKAVHIVNE
PRIFKGIFAIIKPFLKEKIANRFFLHGSDLSSLHTSLPRNILPKE- YGGTAGELDTASWNAVL
LASEDDFVKEFCQPESGCDGLLGQPLLPEGLISDAQCDDS- MRAMKSQLYSCY.
[0231] The amino acid sequence of the human R286 polypeptide is as
follows:
56 MSEERDSLRTSPSVASLSENELPPPPEPPGYVCSLTEDLVTKAREELQEKPEWRLRDVQALRD
(SEQ ID NO:62) MVRKEYPNLSTSLDDAFLLRFLRARKFDYDRALQLLVNYHS-
CRRSWPEVFNNLKPSALKDVLA SGFLTVLPHTDPRGCHVVCIRPDRWIPSNYPITEN-
IRAIYLTLEKLIQSEETQVNGIVILADY KGVSLSKASHFGPFIAKKVIGILQDGFPI-
RIKAVHVVNEPRIFKGIFAIIKPFLKEKIANRFFL
HGSDLNSLHTNLPRSILPKEYGGTAGELDTATWNAVLLASEDDFVKEFCQPVPACDSILG
QTLLPEGLTSDAQCDDSLRAVKSQLYSCY.
[0232] Northern blot and in situ analysis using a sequence from the
R286 clone as a probe revealed the presence R286 mRNA throughout
rat brain. For in situ hybridization, Dig-labeled cRNA probes were
used as described elsewhere (Kuner et al., Science 283:5398
(1999)). Specifically, R286 mRNA expression was the highest in the
cortex and hippocampus while being moderately high in the
cerebellar granule cells, brainstem nuclei, several lateral and
medial thalamic nuclei, olfactory bulb, and striatum. In addition,
this analysis revealed that the expression of the R286 mRNA was
upregulated in response to the multiple MECS treatment. Briefly, a
probe from the 3' untranslated region of R286 was used to hybridize
a Northern blot containing 2 .mu.g polyA.sup.+RNA from hippocampus
from brains of untreated rats as well as rats receiving the
multiple MECS treatment. After one day of exposure using the
phosphoimager FLA2000 (Fuji), an upregulation of R286 mRNA was
detected in the hippocampus (3.72 fold induction) collected four
hours after the last MECS treatment. An additional Northern blot
analysis using 10 .mu.g total RNA from hippocampus from untreated
rats and rats receiving the multiple MECS treatment was performed.
In this experiment, the probe was the ORF of R286 and the level of
expression was found to be induced 2.4 fold in the MECS treated
animals (Table I).
[0233] In addition, rats that developed seizures following
intraperitoneal injection of kainate or PTZ were analyzed for the
expression of R286 mRNA in addition to the mRNA of other IEG clones
(Tables III and V). R286 mRNA expression was observed, by in situ
hybridization, to be mildly upregulated in the hippocampal
pyramidal cell layer, cortex, thalamus, and cerebellar Purkinje
cell layer at 6 hours post-kainate injection. At 6 hours post-PZT
injection, R286 mRNA expression was observed to be mildly
upregulated in these brain structures, while no upregulation was
observed at 20 minutes post-PTZ injection or at 1.5 hours
post-kainate injection.
[0234] Other IEG nucleic acid clones included L073 (concatamer with
Krox-20), L125 (oxoglutarate carrier protein), L201 (concatamer),
R094 (fra2), and R217 (diacylglycerol kinase; accession
#D78588).
OTHER EMBODIMENTS
[0235] It is to be understood that while the invention has been
described in conjunction with the detailed description thereof, the
foregoing description is intended to illustrate and not limit the
scope of the invention, which is defined by the scope of the
appended claims. Other aspects, advantages, and modifications are
within the scope of the following claims.
Sequence CWU 1
1
62 1 527 DNA Eukaryote misc_feature (1)...(527) n = A,T,C or G 1
ttgcagatca gcaccttttg atgatgcctg cccaacagtg ggtaatgctn acagcaaagc
60 accactttac gctttttagt tgtgctgggt tcatggctgg acatacacca
accagccttg 120 accccacagg aatgccaagt tggctggaat gtaacccaac
ctagtttctg cgcttcgctc 180 ctctcccagt gcaaggtgct aaacacccac
tcacaagcct gctgtcaagc tgcgaccttg 240 ggggctggtt agaaagggct
gcctccttcc agcaatagaa gttcatgaat ttgaggctgg 300 agataggtca
agaccactgt gataactata aagactgtag cagccacaaa ggagaccccc 360
aaataactgg aggcatgggc actgacgtac cagatgaggt tatgtttgga gctgaaggct
420 tgctctgtgc ttcttggtag catcttttgt cctcttggga catggttgac
cccatactgt 480 ccactgagct tgggagatga cagttgaata aaaaaaaaaa aaaaaaa
527 2 1485 DNA Eukaryote misc_feature (1)...(1485) n = A,T,C or G 2
cggcttaatt aaccctcact aaagggaaca aaagctggag ctccaccgcg gtggcggccg
60 ctctagaact agtggatccc ccgggctgca ggattctgcg gccgattaag
aagcctgctg 120 atgtccttag gcgaggacat taactccagt ctctgacaga
ctttggacat ccagaataag 180 ttctttttgt atatcagagc acagagccca
gctttagcct ctgatggacc tcaggaacca 240 agaaggaggg acttccttaa
cattctagag atgggactct aactctagct cttgtgttaa 300 gccctgaagt
ccagaaagaa gtagttcttt gacattctag tgccaagatc cagcctctaa 360
gagaactctg atgtctaaag aaagtctttc atagtctagn ccagtcacca gtgaagctaa
420 acacctgaaa actattagat tctctggagc caggaatcca tctcaagtct
ctcataaagc 480 ccaaatgtcc caggagaagt tgacaatata aagccgtatc
tcgatggact tttgaagaag 540 ctcagaaaag gagaccacct tggtagtctt
gatctaggac tctggcttgt ttgtctccag 600 ggacgtttac atgtataaaa
agagggacct ttctgatgat tcagaactgg gactccacct 660 ccatcctttg
atgaaagctc aaatgtccag aaagaggggc ctctctgata ttctagagta 720
ggaccctccc tccagccttt gatggtgtcc agatgtccag aaagaggggc ctctctgatg
780 ttccagacct agggccctcc ctccagcctt tgatggtgtc cagatgtcca
gaaagaggga 840 cttctctgat gttccagacc taagactcta gctccagcct
ttgatgaagc tcagatgttc 900 agaaaggggg gcctccatga tgttctagaa
ccaggactcc acctctagcc tttgatggtg 960 tccagatgtc cagaaagagg
gtcttccatg atttctagga ccaagacttt acctccagcc 1020 ttctatgcct
ccatgtctcc agtaaagctt aggtgtccag aaaagagcat tctcaatgaa 1080
tttatagaac caggactctt tctccagcct ttgatgacgt tcagatgttc ataaagaaga
1140 acttccacaa tgtactaaag ctatgactcc atctccatcc tttgatgaaa
agggacttcc 1200 ttccactctg ttccagaagc ctagctccac ctctaatctt
tgttgatgtc caattatcca 1260 gaaagagggg gcctttagaa caaagactgt
acttttattc attgataaag cacagattcc 1320 agaagcacag aaatctagaa
agagggtcct ccctaacacg ctcgagctag aaccccggtg 1380 caagggtctg
aaacttagac accagaagac cgctttgtcc tacaacaagt ctgcattttc 1440
taaatctcca ggtggctgat cagaagggtc caggaaggta tgggg 1485 3 1854 DNA
Eukaryote misc_feature (1)...(1854) r = G or A y = C or T m = A or
C k = G or T s = G or C w = A or T 3 ggcacgagat cactcagtgt
cttcactgaa ccaaatcgtc atttttacag agagatgcaa 60 agcttcagcg
aagacattta gcttttttaa aatgtataat tcctgtggct acatatgcaa 120
gtagggtccc attatgtttt ttttcattag tggaaactaa tccttttgtg ctgtgtttaa
180 tcagtattag ctttatagaa ttataaatgt atattctact tcttgatcaa
agaacgtagt 240 cgggtattgg ttttagaagt tcaaagtgac actgtatagg
gctttcacgg ttaatgggat 300 tgttagcaaa tcttaaggac atacagccaa
tgattatctg aggttactgg ctaactgttt 360 ttcactgagt tactctgcct
ttttgacatt tttattcttt gtttgtcaga atccagagct 420 tcaggagccc
aaattttttt atwccgtata tatatatata tataaatatc cataagcctg 480
gtggatttgt atgcaatgca ctgcatctat gtattctgat agcatctcat tgatttttgt
540 ttgaaataga aagaaagata gtatcccaaa tgagttatct ttaacagaaa
gctgagttta 600 acttttatta cctatataat aattgatatt gccaattacc
attctgaatt tcatatagta 660 taagttagac attgcttaat ccccttttaa
atgtatttac atagacatga acactcaaat 720 tgctggattt tttaaatata
tctgacataa tttttttcat ctgttacatt caagttagct 780 tgtttagccc
agatttcaga atagtaaagg aggaaaggaa ccgcattcca gggaaacctc 840
tgaggccaag tcagagtcca gaactgtaaa cacacaggcc tgcaagccaa cattagtcgt
900 gaaatcccta acacgtcact ggattctctc tgtcagcgca agtgtcagct
gccaaagaat 960 agacttacat gaagaagtgc ccacatgctg gcaggggctg
gccggctccg gccagcagac 1020 actgctagat tgtaatattt aaggtcgagt
ttcgacctgt ggtacacagc tgtgctgtgc 1080 tcagtcagca acctcagaac
tctgaaaaaa acataaaaaa gaaaaaaaaa aaaaaaaaam 1140 atgcasctgk
ytcacttgtg aatagtgaat gtaaaggaaa gaaaggaaaa ccaaaagctt 1200
gttccatcac aggtatgagc tgctatgatt catgaagaac attccatgga gtatgtttta
1260 aaaccttgtt atatctgaga ggctttaaaa gccaacttaa ctgtttcagg
gcaaccgcgg 1320 tacagacgtg gtctctgtga gacttccacc tgacccaagt
tttaagtggt acgaatgttg 1380 tgcatttaat gttaaggaca gtctgcaata
ataagtaagt agccagcgtg ggtgcccagc 1440 agtgctgaga cctggctgct
ctattgtacg ctttggaaac acaatttatg caacagatgt 1500 ccagatatga
ttctatttat ggaaaaagtt tatatgtttt acaaatggtt ttaccatctt 1560
atattaaatg accttttgac aggtgtgcac tgttttgtct ccagtgagca cataccatgc
1620 ggattttata tgtacatcag tagtgtgaat ccactggcac agtgtgtgta
aatgccagat 1680 gtggtgagat tttatcttgt atatgtgatc agataaaata
actcctgaca gaaactgtaa 1740 ggraacccag ctgaatggtt tgacctggat
grcykrkrtk gtwtggttta tgttaaatgt 1800 atattctttt aatcaatgaa
taaagcatta aaaaatggga aaaaaaaaac tcgt 1854 4 1030 DNA Eukaryote
misc_feature (1)...(1030) r = G or A y = C or T m = A or C s = G or
C w = A or T 4 tctgcggccg cagcatccgg aacaacagga acctccagaa
gtttagtctt tttggagata 60 taagtgtcgt tcagcagcaa ggaagtctgt
ccagcacata cctcagcaga gtagaccctg 120 acggcaagaa gattaagcaa
attcagcagc tgtttgaaga gatactgagc aatagtaggc 180 aactaaaatg
gctgtcctgt gggtttatgc tggaaatagt aaccccatca tcactgtcgt 240
ctctgtctaa ctccattgcc aacaccatgg aacacctgag tttactggac aacaacattc
300 ctggtaacag cacgctcatc accgcagtcg aactagagcg ctttgtaaat
ctgcgctcac 360 ttgccctgga tttctgtgac tttacagctg agatggcgag
agtcctgacc gacagcaacc 420 atgtgccttt gcagcgactg tctcttctgg
tccacaatgc ttcagtgatg ctcaagtcat 480 tagacaacat gccaaacgat
gagcactgga aggccctgtc acgaaagagc tccagcctcc 540 gggtctatct
aatggctttt gatgttaaaa gtgaagacat gctaaagatt ctgaaaccca 600
gtataccact tgagaagggt tcactttgga cagctacgtc acttgtgtct caaggggcta
660 ttggttgatc ttatattcca ggcagtattg accaaggttt cctyaacccm
wtttwtattg 720 atgaatgata tgattgatac gtctggtttt ccggatctta
gtgacaaccg aaatgaagat 780 ccattggttt tattggcatg gcggtgcaca
aagctcactc ttttggcaat tcatggttac 840 accgtgtggg cacacaacct
cattgccatt gctcgtcttc gtggctyttg acctaaaagt 900 gctttggaag
tcaccsraag aaagcattga ttttgaccaa ggtgaactag cccgaccagg 960
aatgtggrwy cccgtacata acctttcttg gagcaggtat tccctggggc cttggtcaag
1020 tcttggcacg 1030 5 1824 DNA Eukaryote misc_feature (1)...(1824)
r = G or A y = C or T k = G or T s = G or C w = A or T d = A, G, or
T; not C n = A,T,C or G 5 tcaaaccnta tctcggtcat tcntttgatt
nataagggat ttksccgatk tccggcntat 60 tggttaaaaa wtgagctgat
ttaacaaaaa tttaacgcga attttaacaa aatattaacg 120 cttacaattt
gccattcgcc attcaggctg cgcaaytgtt gggaagggcn atcggtgcgg 180
gcctcttcgc tattacgcca gctggcgaaa gggggatgtg ctgcaaggcg attaagttgg
240 gtaacgccag ggttttccca gtcacgacgt tgtaaaacga cggccagtga
attgtaatac 300 gactcactat agggcgaatt gggtaccggg ccccccctcg
aggtcgacgg tatcgataag 360 cttgatatcg aattcggcac gagcgaagcc
agggccttgc acttcctagg caagcgctct 420 accactgagc taaatcccca
accccttgtt ttatttttaa agcaaacgag atacataatt 480 tcarccatga
taatttaaga ttatcttgaa ctcttaagga aatgtatata ctaagctatt 540
atagttttta ttttccctaa ttcagtggca taatacctta ccttgagtcg tttactactt
600 tctttggttt ctaaaaactc tactgctaaa ttacaatgta aaaacatagg
gctcgtatat 660 actgtagagt gctgtagatg tcctcgtcat caactatgca
ataacagtct gatcgacaca 720 tttcaggakc gatcactctt tggtgtgctt
ctttaaatac tttcagaagc ttaggatgtg 780 caaagcagga agactgtggg
tgtaaatgtt tacttatttc tttgagagtg ttagtaagtc 840 ttttcdaaat
tgcttttctc ttcaaaatta tcgttaactt aaatgataat tatctttgag 900
gttaaacaga agctcattga caaactaaag tgacttttta gggcattctt tgagatcata
960 gtcttatatc ttggggacta aaatgtcatt agaccctaat agactaactt
gtatgtttgt 1020 gtggggaaac gttttcctct ctcattcaag gtaactgttt
gctgcctgtt gttacttgtg 1080 tagcattcta gaaaatggct aggtttttta
taagatttaa gacaatagaa gtagttttat 1140 attattatag ttctgttgga
atgtgatcct gaaattatta ctgaaaatta gaatttttat 1200 ttcgctaatg
acaaccttga ctctcagaga tgcagtgtaa attgatacct catctttccg 1260
agagttcaga gcacagggcg gcagtatgtg aagctgcttt tgcactgacg cattttgata
1320 agtttggcta ctgtaatggt aaaaggctcc tcaggcactg actgcatttg
ggttcttccg 1380 atgggggatg atccgttctc gtggtgctgc tggacttatg
cattttggag gtactgcatg 1440 tatcttccac actgcttgac attttctctg
atctgtgtgt ttgcaccaac tcattaaaag 1500 aaatatgcag aaatatcttc
taattcgttg atcttcgctg tatgacagtt ataatattaa 1560 acacttgggt
tgatccactc tgtttacatt tatctttcta agcgtcagaa agggactaac 1620
ttgaaattat atctagaggc tttgtatcat ttcaaaaatt aaatttcctt ggatacttta
1680 ggcaatatct taaacaactt tttaataaat ttaaatattt atatttacgt
aagctaaaat 1740 atacatgaat gtgcttttta ataaattaaa tacagtttat
acttatttgc caattcacaa 1800 ataaaaaaaa aaaaaaaaaa aaaa 1824 6 1230
DNA Eukaryote misc_feature (1)...(1230) r = A or G m = A or C s = G
or C w = A or T 6 tttttttttt tttttttttt aargggrcca ccccaccgsg
ctaaaggccc aggggccccc 60 cccttggagm cccaggggtt ttggcccmcc
ccctcaccca aatggtctgc caatgaccca 120 ggtactcaca acatgttcca
ggaggagmct ggggccagga ttttgaccag agggtatggg 180 aagggaaagg
ggagaagaaa tcgacattta tttttattat ttattttaaa tgtttacawt 240
ttctttgtgt tgttccaagc cctgaataga aacagatagc attaaaggac tctgttccca
300 ccccttctct gtctctctct cccccacttg tgctaactta ggataacact
ctctatttcg 360 ttttgtttct aaagtgattt gtggacttgt gccgtgtgaa
ctgcattaaa aaggttctgt 420 tttcaaagat cgattgtcgt tcctgtgggg
acagtggctc ctaagaaatc tgcattgtag 480 gagaagacaa tgaaagaccc
tggccctgtc tctcaaaact taactctctg tatgatttaa 540 aaaaaaattc
catttacttt actttgtggt tacttgattt tgaggaagaa aatattcaac 600
tttgtataaa gactaggtat cagggtttct tttgcagtgg gagttgtata tatatcgtat
660 tttggtatat cgtagaaact caagctttat gcatccgtat ttgggatatg
tcaatgacgt 720 gcagtgaaat ttgctattag accctggagg caaacgagtt
gtacaaggtt ttatggctcc 780 atggggaatt ctaatttcct ttctggggac
cttttgtccc gtttttacag taatggtgaa 840 atggtcctag gagggtctct
ctagtcgaat tctccaggca ggaccacgtg ctcaaaaaat 900 ctttgtatag
ttttaaattt ttgaggagta tctctgctca gaagcatctg tggtggtgtg 960
tgttgcgttg ttctgtgtac tgtgtgtgac acaagcctac agtatttgca ctaaggaaag
1020 ctgtttagag cttgctgcta tggagggaag aacatattaa aacttatttt
ccctcggggw 1080 ttrtwcwmgt tttatgtwct tgttgtcttg ttggctttcc
tactttccac tgagtagcat 1140 tttgtagaat aaaatgaatt aagatcagmw
rwrwrmaaaa aaaaaaaaaa aaaaaaaaaa 1200 aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa 1230 7 1516 DNA Eukaryote misc_feature (1)...(1516) n =
A,T,C or G 7 tcaggcctna gcaatcctcn ttaantttga nccaagntta actcttgggg
cgaattcctg 60 tgnttgcttt ctttccccat anttccaggc ccacaaangg
tttctgtgan tccgagaatc 120 ggcccaccat gcagacccac ngagaggatt
cagaatgtgt gtgagagtga gtgtgtgagt 180 gcgcgtgcgt gtgctttgta
tgtgtgttta tagatgtagg acattaagtt ccttctgaca 240 cagggaagat
gtgagaagga tggcctgaca tcagatgaca agaggtctta tagcacatct 300
ctgggctttt ccctacccag agaagagccc cctttgatac aaatcagttg gattttcata
360 tgcttcaaag gcttgatctg tgagtcactc cagtttggga cataggtctg
tctgtggctt 420 tgagaaaagg tactttcaaa agagggcttt ccagagcaca
gctcacagcc agctgttagg 480 accccaccct tctcctttat tgtggaggtg
actcacagca gactgacagt ggtcagactg 540 agctttctgc taaggtggtg
aggtagccaa cactggcatg tctcggtagt ggtttgggca 600 aatttccgca
ggtctcttcc cccaaccctg cctctgatga ataaagacaa tgagtacagt 660
tccttaattc aggcttttgt gactagctta ctacggaacc gaaaatggtc ccctttgtac
720 aagccgagct gttatggaat cacggtgaac cagacccagg tctgtggcac
ctgtttgttt 780 tttttttttt tttttttttt ttagctctca tttctacggc
atgctttcca aggaaccaaa 840 ggagggtctc agagatgccc caaacatccc
aaagtacaca aagctaagta atcgattgct 900 tacttattgc acagctagac
acggatttta agtctatctt aaagctttga agcaagctta 960 gcttctcaaa
ggcctagcag agccttggca ccccaggatc ctttctgtag gctaattcct 1020
cttatccagc ggcatatgga gtatccttat tgctaaagag gattctggct cctttaagga
1080 agtttgattt ctgattcaga gtccttgttt ccctgacttg ctctgccagc
cctgcaccag 1140 ctttttcgaa gtgcactatg cttgtgttta acttctccca
gttttatttg ggcataaaag 1200 ttgttgcctt tatttgtaaa gctgttataa
atatatatta tataaatata tgacaaagga 1260 aaatgtttca gatgtctatt
tgtataatta cttgatctac acagtgagga aaaaaatgaa 1320 tgtatttctg
tttttgaaga gaataatttt tttctctagg gagaggagag gttacagtgt 1380
ttatattttg aaaccttcct gaaggtgtga aattgtaaat atttttatct aagtaaatgt
1440 taagtagttg ttttaaaaag acttaataaa ataagctttt tcctgtgaaa
aaaaaaaaaa 1500 aaaaaaaaaa aaaaaa 1516 8 1534 DNA Eukaryote
misc_feature (1)...(1534) n = A,T,C or G 8 gtggcccctg ctcgccgcat
catggagcgg atccccagcg cgcaaccacc tcctacctgc 60 ctgcccaaaa
cgccagggct ggagcacgga gacctgtcag ggatggattt tgcccacatg 120
taccaagtgt acaagtccag gcggggaata aaacggagcg aggacagcaa ggaaacttac
180 aaattgccgc accggttgat tgagaaaaag agacgtgacc ggattaacga
gtgcattgcc 240 cagctgaagg atctcctacc cgaacatctc aaacttacta
ctttgggtca cttggagaaa 300 gcagtggttc tcgagctgac gctgaagcac
gtgaaagcat tgacaaacct aattgatcag 360 cagcagcaga aaatcatggc
cctgcagagc ggtttacaag ctggtgatct gtcgggaaga 420 aatattgagg
caggacaaga aatgttctgc tccggtttcc agacctgtgc ccgggaggta 480
cttcagtacc tggccaagca tgaaaacact agggacctga agtcttccca gcttgtcact
540 catctccacc gtgtggtctc tgaactcctg cagggtagtg cttccaggaa
accattggac 600 tcagctccca aacccgtgga cttcaaagag aagcccagct
tcctagccaa gggatcagaa 660 ggccctggga aaaactgtgt gccagtcatc
cagaggactt ttgctccctc gggcggggag 720 cagagtggta gtgacacgga
cacagacagt ggctacggag gcgaattgga gaagggtgac 780 ttgcgcagtg
agcaacccta cttcaagagc gatcacggac gcaggttcac cgtgggagaa 840
cgcgtcagca caattaagca agaatctgaa gagcccccca ccaaaaagag ccgaatgcag
900 ctctcagatg aggaaggcca cttcgtgggc agtgacctga tgggttcccc
atttcttggg 960 cctcacccac atcagcctcc cttttgcctg cccttctatc
tcatcccacc atcggccact 1020 gcctatctgc ctatgctgga gaaatgctgg
tatccgacct ctgtgccact gttataccca 1080 agcctcaaca cctcagcagc
agccctctcc agcttcatga accagacaag atccaactcc 1140 cttgctctgc
ccagaaatcc cttctccctt ggcacattcg tcccttgact ctcaagcctg 1200
ctcaagccct gaagcagatc cctccttaaa cttagaaaca aagataaacc ttgagggcaa
1260 tcnctgcgcc ttgctttcct tcccacaatt caagacacaa aaggtctgta
ctcaaaacag 1320 agagatcagc ccaccctgca gacccacaga gaagattcag
agtgtgtgtg agagtgagtg 1380 agtgtgcgtg cgtgcgtgct tgtatgtatg
tttgtatatg taggacaata agttccttct 1440 gacacaaggg agacacgaga
aggatagcct gacatcagat gacagactgg aggactgtag 1500 cacatctctg
ggcgtttccc tacccagaga agag 1534 9 5470 DNA Eukaryote 9 gcacgaggga
gtttatttcc acgtctctta ggaaagcctc gcttggttac acatggcaat 60
gattgcaagc agatacacgt cttaacacca gagtacagta cacacacatt gagctgccct
120 cgtctaacaa gcagttgcag tttgtttaaa tgtgaatatc tatgaaacga
gcaaagcaac 180 tttccagagt atagcttatc acagaatagt aacacatggg
ccgctactgt atcatacaga 240 gtacaactct atagcttttc atccccgtgt
gagcatttcc aaatcactca atgagcacca 300 agcacggaca agtgactaaa
aaggctagtc ccaatctccc cgcaaccctc ggcggtaagg 360 gtaaagaatt
ttgtttcaag taagttttct cctcgtctct ctcttctgaa gacctgagca 420
aaaccaacat tctaaaccac cccaagatat gatactagaa tttaaaggcc cgatggcttc
480 aacccagaac cttaacctac tagataaaat ctctccgaat ctgactcact
gatgctgtta 540 agtccgacag tacaatcaca tagtacctct ttgatactgt
caaagttggt tttaaaaatg 600 ccctaagaaa accaaatcat ttttgggaga
tgttctaagc aagctttcca acatataaag 660 aacaaaacca tgttactaaa
aacatggtgc aggtcctcac aaaacattta ctgctactac 720 caggaaacca
agctactctt ggtttgtgct cctggtgata actggtgagc tttggacagc 780
tgctggcaca tgtccactgt gttccgtttt ataatcaagt gtcagttttc cactcgacag
840 agattaaaga caatagctta aaagtgaaaa tgaaatttca agtagaagct
acaattgaat 900 gctacttgtt gagactttta actttcacat ccaaatatca
aaaacttaac tttgacgaca 960 catgcacaca aacacaccat ttgggaaagg
gtcttgttat gcagttcaag ctggccttga 1020 actcatgatc tcctgcctca
gtttcttggg cagtagcact ggaccttact gtgggcagaa 1080 agtattgctc
caattagaaa gcattactat acacttcact tcgtcatgtg cctagtgtgg 1140
ctctgaaggc ataggaacaa tgaaattaaa ttcttcagca gctgaggatt ctctatactt
1200 caacattctg aacttcaatc atggcttcac atttgaggct gagctagata
caaaaatatc 1260 aaaacatccc atagaattgt ttatttccct atgttactgt
ttacccaagg aatgtgaaga 1320 ctaaaaagga ctcatttggt tgtttaatta
tgattaaatt atgtaaatat acaaacattt 1380 aacaaagcca tcatattcca
atcttttacg aattctaact gctagcagtt gagcagcttt 1440 tagatatcac
taataaaata tacaatttaa aatagtcgca ttcaatccta ctaactttat 1500
aaataacttc ttaggttaga cttcttcctg cctaagttta taagacagtc taaacccaaa
1560 actcaacaca tattaagctt tttaaaaact ccatatagtt ctaaagtaac
ctcaatgtat 1620 tcccaagaac cgccaccatc aatcagctca ctccctcaca
ccactgactt taagacgctc 1680 ctgggtggag aactgccagg cagaagctct
acctttctag tgtgtgtggt ggtctgctgc 1740 tcctagtcca gatctggacc
acatcagcac agcatcagtg tgactcagca ctgaggcctt 1800 gagcgctctt
ccccccgatg gcctgtgtat agaggtgtct aattccttgt gtatagatgg 1860
cctgtatata gaggtgtcta attccttggc tctgtatgta taggtaatgt gatactttac
1920 cattaaagca ctattttctc cattcaagaa tttagtgata taggaaaatg
agtggacttg 1980 cgagactcag aaaaacaaaa cataacctgt cttgaattca
aaacaaacca tgggtgtagg 2040 ggggaactga tgaaagttta tgggtttaac
tctaggtaat taactaagac agtcacgaaa 2100 cacattatca aaatcctttc
aggcccagag cttgtactgt accccactgt gagaccacat 2160 cacaaccccg
gattgagctt tatccacaac acctacacca tagtaacgca aagtgcacaa 2220
tgtactaaaa taaattccta ttagttttat gcaaactatg gtataaaatt atcacctgcc
2280 atacatattt tgccatggca ccaacttcat ataataagcc aacgtataat
caaagtcctt 2340 accagcacca atcaatgtcc ttggcaccac tggacactca
ccgtcaagct gttcatctaa 2400 gagccagtct gttctgacct gaacagttgt
gcattccacc ttaccacacc caagtctgtg 2460 agccggacaa gtgtttaaat
gcagttttac atctaacggt gcaggttaag ccgagcactt 2520 gaaactgatc
actcattaat acctgtctcc ctccatacat gtacaccaca tgtacacaga 2580
actatgtgct ctgacttcag aatagctctt cctgttggca aaacaccaca gacatgaagg
2640 ggcctagtgt gaagcgagct cacagaatgt tggatggaac ttcgactata
atggaaacac 2700 ctgcaaaagc tttgctaacc cagcaaacac tcaacactta
ccaaagacaa cagggaagtt 2760 aaagttagct cgccaagaga tgggctgggg
aggtgggggt gtaactcaaa gaaagcttta 2820 gctaacaaaa acgaatgatg
gacaacttca gaaattccct aaaaacagaa cctgaaagtg 2880 caggtgaggt
tttgtccttc agtaacaaat gcagacagat tcccaacagg aataaaacag 2940
tctggggctt tgaaacctgc tagatggaaa cacgaactca aaatgtggaa ccaaggaaaa
3000 ccaaatactt aaatgtgtaa gataatttat aatagtaaaa agttgcaaat
tgctgtgact 3060 tgatttgccg aaaacatctg taaatccaca ctggcagtta
gaagaccagt
tcccacatta 3120 actcctctct cagcaggtaa ccgtttgtgc gcagaagtat
ctgaaacatc gcactactgc 3180 ttattttatg gtgtattgtg cagaatctgt
acatgctatt acagacaata catatttgta 3240 aacctggtca tgcaaaatca
gtgtgtacaa ggggatattg ttaagcctta taaagtggta 3300 ctttattatc
tttgtgacga tgccaatctc tccgaaatat agcatatctt aaatggatat 3360
tctttatctg ccagttaaaa tcattttatg tcactgaaag aagaggttat acaaggaaag
3420 aaacatggtc cttgtgttgc agaattgatt ttaaatgaga gaatttacaa
aaccaagaaa 3480 tccatggtca taaagtttta acattttaat cctacacatt
acagggcaaa cagatactgg 3540 accctatttc cacattccat aaatccaaac
tttagttccc atttcaaacg ttgccctaac 3600 cactaaaacc atcagtggtc
ttacaacctc tggattatgg aaatacagat ttctgaagta 3660 aaagctacaa
aaacaacaat ggaagaaagc tgaacaaact tcccatgaat gaaaataaaa 3720
gtggaacatc ctgaagctct agacacttct ctcccgtgtc tatggtcaac ttgtcggttc
3780 agtgcactgt gcggtcaaat gtaatggtcc tcatgtggaa cacacgtcta
actagtgtcc 3840 attgattcca agttagtgga cgaagaatct ttctggatac
tttcaaagat ggctgccagc 3900 tccgggttgg agctgatctg tgactggaac
tcactcatga gagggctctt ctctgcctct 3960 ggaatggtga gcagtgcagc
tactgccctc atggccgagc gctttaactc gtcctgcttt 4020 tcaaactcct
gctttacaga gttcgccttc accttagttg tacacgtagc tcgtagtggc 4080
tcaacaagcc ggtccaacct ctgtagtact gcacttggac aaagggtaga tagtctcacc
4140 aacattaaaa atgttagcat cttaatatca taatggtcct tcaaaccatc
ttccacatga 4200 tttagaaatt caaagatatc cagtctgtca agacagctgt
ctagaagtgt gtacatacac 4260 tcaaaagctg cctttctaat gtccaggccg
tcatcaaccg tgtgcttaaa cgggcccatc 4320 tctacctctc ttataagttc
cttcctaact tttgtctcat tgtaaagatg tggaagaaca 4380 gaatccagaa
ggtcccgtat cagtgacggc ttgttatggg ctgcagaatt gaatgtgacc 4440
aaggccactc ttcttacatt caaatctggg tcttccaacg tttttagaaa atcacctatg
4500 cagttcttga gcagtggatc ttcttgacat cttgaataaa ctgacctact
acagccggtc 4560 cctctttagg gcatgctcga gtaagggcag ctacacattt
ggcaatggaa tagtaagact 4620 gcttatgagt aagagctgtg ctctgagagt
aaactggacc cgttagcatg cgcagcaaat 4680 ccatgtatcc tagattgttt
gttccagtga caaccaaagc ttggaagaag tctagcatgg 4740 cactaagagc
tcctccctgc agcagaggtg accttacaag tccaatcagt tcattgagaa 4800
tagatccgct tatctttgaa agggaggagg gatatacttt tgccagggta gtaaggaagc
4860 tgatagccat ctgggacacg tgcatatcac tttcgctgat aagaggaggg
agctcatcca 4920 gaactgcatc aatcatggcg gccgtcaaac tgtcactata
gtttttaatg agaatatcta 4980 gggcagagag ggtccccagt ttcaaagctc
tctgattttt cctgagaaat gaagcaagga 5040 tagggactcc ctctcccagc
acaggcctca gatctatctt caaaggtgac ccagcaatca 5100 gggtcagtgc
tttcactgtc gttagccggg tgatttcatt cttgagtctc tccaagaaaa 5160
tctgaagtgt atttgataag tcagggccca aattgtctcc aagattgcaa ataatctgtc
5220 ccatacagga aatagccctc tccttgactt cctgatcaat gtcagctgct
tttaagcgct 5280 taattgtaca agtgaagaga tctttgatgt aaggcgttgc
atcgaaggag gagggttggt 5340 ccagaggacg gattactttg acaagctgct
gagtgacaag aagggcttct gatgtgatct 5400 tgtaaaatgg gtcaccaaca
caagccacca ctggagggac caaagcctga acatgcgggt 5460 ggaaaacttg 5470 10
2515 DNA Eukaryote CDS (414)...(1055) 10 tcgccgcccg aagtcgcgca
gcttccctgg cgaacgcgga agcccgaaga gcgccgtcct 60 cgggccctgt
cggcgctcag gccccttcgc gcgcctcctc gctcggccgg gacgttgctg 120
tggaggcgtg aggcgccggc ggtcgagcac ctggagcgac ggtagcccgc ggcctgcggt
180 tcttctcctc ccccgccgcc ctcccacccg agctgcggcg gggctcggcc
gcctcggtgc 240 ttctgcacga acaaaggagg cccccgcggc gccggcgcag
ctccatctgc ggtccgatcc 300 acccgggccc gcggcggccg ctagccagcc
cttcccggag gcctcagccc ggcccaccgc 360 ccggcgtcgc gcgccagctc
gctagtgcat ccgggccccg caggcacaaa aat atg 416 Met 1 gct cag gag act
aac cag acc cca ggg ccc atg ctg tgt agt act gga 464 Ala Gln Glu Thr
Asn Gln Thr Pro Gly Pro Met Leu Cys Ser Thr Gly 5 10 15 tgt ggc ttt
tat ggg aat cct agg aca aat gga atg tgt tct gtt tgc 512 Cys Gly Phe
Tyr Gly Asn Pro Arg Thr Asn Gly Met Cys Ser Val Cys 20 25 30 tac
aaa gaa cat ctt cag aga cag cag aat agt ggc aga atg agc cca 560 Tyr
Lys Glu His Leu Gln Arg Gln Gln Asn Ser Gly Arg Met Ser Pro 35 40
45 atg ggg aca gct agt ggt tcc aac agt cct acc tca gac tct gcg tct
608 Met Gly Thr Ala Ser Gly Ser Asn Ser Pro Thr Ser Asp Ser Ala Ser
50 55 60 65 gta caa aga gca gat gct act tta aac aac tgt gaa ggt gct
gct ggc 656 Val Gln Arg Ala Asp Ala Thr Leu Asn Asn Cys Glu Gly Ala
Ala Gly 70 75 80 agc aca tct gaa aaa tca aga aat gtg cct gtg gct
gcc ttg cct gta 704 Ser Thr Ser Glu Lys Ser Arg Asn Val Pro Val Ala
Ala Leu Pro Val 85 90 95 act caa caa atg aca gaa atg agc att tca
aga gag gac aaa ata acc 752 Thr Gln Gln Met Thr Glu Met Ser Ile Ser
Arg Glu Asp Lys Ile Thr 100 105 110 tcc ccg aaa aca gag gtg tca gag
cca gtt gtc act cag ccc agt cca 800 Ser Pro Lys Thr Glu Val Ser Glu
Pro Val Val Thr Gln Pro Ser Pro 115 120 125 tca gtt tct cag ccc agt
tct tct caa agt gaa gaa aaa gct cct gag 848 Ser Val Ser Gln Pro Ser
Ser Ser Gln Ser Glu Glu Lys Ala Pro Glu 130 135 140 145 ttg ccc aaa
cca aag aag aac aga tgt ttt atg tgt aga aag aaa gtt 896 Leu Pro Lys
Pro Lys Lys Asn Arg Cys Phe Met Cys Arg Lys Lys Val 150 155 160 ggc
ctt aca ggg ttt gac tgc cga tgt gga aat ttg ttt tgt gga ctt 944 Gly
Leu Thr Gly Phe Asp Cys Arg Cys Gly Asn Leu Phe Cys Gly Leu 165 170
175 cac cgt tac tct gac aag cac aac tgt cct tat gat tac aaa gca gaa
992 His Arg Tyr Ser Asp Lys His Asn Cys Pro Tyr Asp Tyr Lys Ala Glu
180 185 190 gct gca gca aaa atc aga aaa gaa aat cca gtt gtt gtg gct
gaa aaa 1040 Ala Ala Ala Lys Ile Arg Lys Glu Asn Pro Val Val Val
Ala Glu Lys 195 200 205 atc cag aga ata taa aattactaca tgtgaagaga
ctgaaacttt gtttttattt 1095 Ile Gln Arg Ile * 210 taatatatcg
taggaaaaca ttaaagagca gatgcatggc cattttcctt tgatgttctc 1155
cagagttttg ctttatactt gtctgtcata taattgatat tttaggatgt ttgggtgttt
1215 gttacaggca gaattggata gatacagccc aacaaatgta tatgccctcc
cctcagtaaa 1275 attggacaaa aatatgcaca gcaaattgaa atacacatat
actaggaaca aaatttagtt 1335 ccatgtgcca aactgaatga aatctctgca
tgtttgcagc atatctgcct tttgggaatg 1395 taatcaaggt ataatctttg
gctagtgtta tgtgcctgta ctttaaaaaa atggtacacc 1455 agaaaaggac
tggcagtcta ctaccatagt caaacttcac cttaatttcg acatgctttt 1515
ggaagcagga agaaagctac aaaaccagta tttggtgcca tgtgtgagcc tggttaaatt
1575 ggtcttctaa aagctgtcaa ttaggacatt ctgcgaaagg taacatcaca
actggttctg 1635 agtaaaacca tcaagtcaac agcagggtgc ctgagataat
ctttgaagct tattgtgctg 1695 gcctgcacca gaagatatct gcattctcat
tactaaaatt gtagcacaga actgcactag 1755 gatttgttta caagaagaaa
ttaaaactct acgtttggtt ttcacatata gcagctctgt 1815 taaataacat
gcatctgaat tttaagttgc aaaggtatct gagcagttag tttttcatgt 1875
gcatcttttg ttgaatgttt tggttcaaga aagaatgttt aaagcttttt aaagacttca
1935 gttcttaatg taactgtacc cttctgcatg gaaaatcata accaacatgg
ctgcagtaga 1995 cttctttagt ggtatccagc accacttgca gagggctgct
ttatcatatt gtatttgggt 2055 gtaggactct agtgttcttg ggtgtattgc
atgggctgca ttatctacag cattgtacaa 2115 taacaactag aaaaggcagt
atacttcact gatgcttgtc tggtaatatc acttctgtgt 2175 tataatggaa
ggttttttgt gatgtatgaa acttgtgttt tttatatata aatgagtata 2235
gttagattag tgttgtggta atgcctgttt tcatctgtaa atagttaagt atgtacacaa
2295 ggcactactt ctgatttatt gcagtgttca gtcctagttt ttctttatta
aaacattcag 2355 ttttgcttca attttatgta ctttagttct aagttagatt
tgcagatgtg tacagatagt 2415 tcatatttat gtattgcaca taatcatgct
attcagcatt gatgctatat tgtattatgt 2475 aaataataaa agcagtgtac
agagggaaaa aaaaactcgt 2515 11 213 PRT Eukaryote 11 Met Ala Gln Glu
Thr Asn Gln Thr Pro Gly Pro Met Leu Cys Ser Thr 1 5 10 15 Gly Cys
Gly Phe Tyr Gly Asn Pro Arg Thr Asn Gly Met Cys Ser Val 20 25 30
Cys Tyr Lys Glu His Leu Gln Arg Gln Gln Asn Ser Gly Arg Met Ser 35
40 45 Pro Met Gly Thr Ala Ser Gly Ser Asn Ser Pro Thr Ser Asp Ser
Ala 50 55 60 Ser Val Gln Arg Ala Asp Ala Thr Leu Asn Asn Cys Glu
Gly Ala Ala 65 70 75 80 Gly Ser Thr Ser Glu Lys Ser Arg Asn Val Pro
Val Ala Ala Leu Pro 85 90 95 Val Thr Gln Gln Met Thr Glu Met Ser
Ile Ser Arg Glu Asp Lys Ile 100 105 110 Thr Ser Pro Lys Thr Glu Val
Ser Glu Pro Val Val Thr Gln Pro Ser 115 120 125 Pro Ser Val Ser Gln
Pro Ser Ser Ser Gln Ser Glu Glu Lys Ala Pro 130 135 140 Glu Leu Pro
Lys Pro Lys Lys Asn Arg Cys Phe Met Cys Arg Lys Lys 145 150 155 160
Val Gly Leu Thr Gly Phe Asp Cys Arg Cys Gly Asn Leu Phe Cys Gly 165
170 175 Leu His Arg Tyr Ser Asp Lys His Asn Cys Pro Tyr Asp Tyr Lys
Ala 180 185 190 Glu Ala Ala Ala Lys Ile Arg Lys Glu Asn Pro Val Val
Val Ala Glu 195 200 205 Lys Ile Gln Arg Ile 210 12 660 DNA
Eukaryote misc_feature (1)...(660) n = A, T, G, or C 12 attccaaaaa
tgcatagatt acaaagaaac accagacaag ctcaaactca aggatattct 60
acaaataaac cagtaccttc aaaatgccat gctaccaggt acagacaggc gaganactgt
120 tccacactga ggaaactaac aaagtatcca tgaagtccat aattgtgggt
caaatccagg 180 acctgcaaag gggatttggg gataattttc aaaatttgac
taaggtctgc agagtagaga 240 gacgaggtca atgccaatgt cctgattttg
acagtaagta tttaaatatg caggagaaca 300 acctaaccaa gaggctgcca
acacacttcc tggctgtggc acaactagat ttaaaaccag 360 caatttgttg
gttcttgttc tcaaatatca gttacctgca agcactccat cgtgaaagga 420
ttgagagcat gaggtgatgt gttgatggtg aaaatgagaa ctgactgagc acaggaaaga
480 gtggcatgat gggcagggaa aggggagaca aaggtcacaa gagcatgcaa
cactcagtga 540 actacaggac actccaaaag gcactctgct gtctagcttg
gatctggagg aggatcagnt 600 attaataagg gccctggaag ggncaaagct
agcctcccag ctgctggctt cccatctgct 660 13 1475 DNA Eukaryote 13
gccaccacca ttgttaatgg agggaggctc tcccttgtta tttctcagaa gactgaatgt
60 ctgtaccaaa aggctcatgg ctttctctgg gcctttccat ttaaggttat
agttttttat 120 gtagtgttac taaaatctag gcttgttact aaagtgggct
ttgtagttat tggtatcggt 180 ggatttttat gttacttgga gtccagaaca
gggagagctc accacaaacc tctcctttcc 240 ctggaccaaa caccctctct
gtcctgtgaa ctcacctttt cttctctgtg ggtcactccc 300 attaccacac
tggtgagcga gccaaatgga tgagagacac aaagaccgta gttcttgaga 360
gacattattt ttttcaactt tgttttttaa gagattttat gtgttgattt gttttggttt
420 ggtttaaagg gattcatagc taacttggat ttttgttacc tcagctctgg
gagaggattt 480 ttgctgaatg actattaatt acctgagcat tgttgctctg
aggtcatggc atgctagcct 540 atgtctgtta cagtctcagg ctgcccttgt
ttcctcgttc ctgtgctatt gtgctacacg 600 ctcaaggggc cttgactctg
cttacacaca ttaggggcag tgtgagtaaa tgtgcagtgt 660 ccacacttga
ggacatgaat gtctgcactg tcactttgct ctgggtgtga agtccctggt 720
ccccttgctc ctgtagcttt cttttgatcg actactggaa ctcaaccctg tgtacaagag
780 cagcactgcc tctggtgggt ggtgtttgca gccaggatta gatgccagtc
ctcgggttcc 840 ctggccttgt tggaaaggtg tgcttccttg aggtctgaga
atggaaggct ctgcctcact 900 ctagctagga ggcgcaatgg gaaagtatga
gttcagggcg tcagggcagt ggctcctgaa 960 gagccagctg tggacagagg
gagtgaggct ttatttaaag tgacaggaag aaacatggcg 1020 ttttggtata
ttgggagcaa tgccaagatt ccctcctgcc ctacataggt cacagacacc 1080
ttcccaacca tcccctcctc cacttccata aatgaagaca gccctgatga ccctcacccc
1140 ttttgcatag gtcactggat cccactgtcc ttcctcggtg cttacacact
ttacagaccc 1200 tttaggcgag cccttgcata gagcgttatc tcagtgctcc
attccagtcc tgactccctg 1260 tggccattga gactttggat ttaagaactc
acattgctag ggagaggggc tttgctggga 1320 aaggtgactc ctctgtaacc
tagcctcttg tgctcctcca tgacagaaat gctgggtgga 1380 gttttacatt
tgccaatggc cagcttgtga atatcttcat atacactttc tattcatgtt 1440
actgtagttt ctgttttgaa ataaaacttc tgaat 1475 14 953 DNA Eukaryote
misc_feature (1)...(953) r = A or G y = C or T 14 catataaatg
tactttattg ttttaaacag aacgaaagaa gaggcagaaa acatttgcat 60
gtaagtccta gcttataaat gtagttttta gtggtggcat ctctaacacg tcgttcaggg
120 actgtttcct tttgcctcct tgtactgtga gcactgacac ttgagaaaag
cacatctggc 180 ggacatatgt ctccagaact ggaagaactt ggagagcaaa
catttttctt aattcctcta 240 agtaatcttt agtaaaacaa aagatgatct
ttggcataga ttcatacttt aaaggcattg 300 atatgcattt atatcaggta
agcaactata cagatctgct gagagctttc aaaagaatct 360 gttatcagct
gaaaggaaat aggggaagcc tgagtattca gggtcaactt aagatttgca 420
agttcagtgt tggggtcaac atactagatg tgggaagaac atccaggcaa ggtcttagtc
480 ctgtattcac ctggttcttg atttctggaa gaagcatcca tgcgctagga
aatgcttata 540 cagccgaggt aaatgcaaaa atgagtaaag tcactttttc
actaactttg cccaataggr 600 aacatgcctt tctgataagt agataccata
ctctttattc ttgaatactt tatattgaga 660 gaaggttgta gttggttaaa
agcaactggg aactataact tcctactgat ttttccctag 720 cagcaccaga
attatattct gcaaatgcta ttctccctta cataggaaat atccttcaga 780
caaaattgcc tttccattca gtctcttaag agyttaattt tgaatggact tttcaaagtt
840 acaagcaaag tcaagtgtgg tggtaggagc taagaggctg acacaagtag
atgacttgaa 900 tccagaagtt caagactagc ctggacaaca tagagagacc
cagtctcaaa att 953 15 911 DNA Eukaryote misc_feature (1)...(911) n
= A, T, C, or G 15 ggcggggatc tctcggctgg taagaagggg cagtggtacc
angcgggcac ttattcagtg 60 tgccaaggat atcgccaagg cctctgatga
ggtgacgagg ttggccaagg aggttgccaa 120 gcagtgcaca gataangcgg
nttagaacca atctcttaca ggtctgtgag cgaatcccaa 180 ctataagcac
ccagctcaaa atcctgtcca cagtgaaggc caccatgctg ggccggacca 240
acatcagtga cgaggagtct gagcaggcca cagagatgct ggttcataat gcccagaacc
300 tcatgcagtc tgtgnaagag actgtgcgag aggccgaagc tgcttcaatc
aagattcgan 360 cagacgccgg atttactctg cgctgggtca gaaagactcc
ctggtaccag taggcacctg 420 gtcagacctg gctggtacac agacctctgc
taatgangan gtgaccatct tgagcttcag 480 aagccattca gagttgccaa
ggggtggnaa atcaatccct ggtttcacac accaagaaag 540 ggaatggggc
ctccttcaca ttagaataaa catttatact cttgtcatgg gacactttga 600
aagtgtctct cctacaaaac ccctggtacc tttcaggntt actccnggtn gcaanntcct
660 cccccaaggg gaatttttta ccaataaaag gctcaaggaa ttaanggcgn
ttgaaaacca 720 acntnatcca angggaaang cccccntggc cttctggccc
ccttgggggn acaatttttc 780 ntcccnctgg gtgttttaaa tggggtttca
accttggggc tggncctttt tccncccccc 840 cttttaaggg gcttcctccg
aaggaacctn agaaaacttn aagggccaaa gntccanttt 900 acnaataact g 911 16
621 DNA Eukaryote 16 tttttttttt ttttttttcc tcccttaaaa gataaactaa
taaactcttc aatggtcttt 60 tcagtatagt tcttatgtag tttaacatag
cttataaatt gagtttaaca ataaactcaa 120 gaagataatt ttataaaccc
tgttttccaa tctgtcattt acttaaatta ttttggttgt 180 tttccctttt
tttccttctt tctcaccccc tccctctcca tgaagattca ggtgcttaac 240
atatcatttt tttccctgct ggaattttag cattgatatg aaccatggac aagtatattc
300 tgctgccaca aagactgtaa agtgcttcat ttcaacagct gaggcaagcc
aagtgatcat 360 taataaagct tttcttgctt ccttcagtgg tgttggtagt
aaaatggtag gtaaaagtta 420 ggctgcaagt tcaataaatg agatttacct
atcattccac ccttgtgtat tcattcacct 480 atcctggttc aagcagtttg
agtcaactag gcatttaaag gcattgtgtt tattacttta 540 tggttccaac
tttacatact tgtcagggat gaagtctgat aggttaagga cagtagaaat 600
ttctgtgcaa caagcagcaa c 621 17 567 DNA Eukaryote misc_feature
(1)...(567) n = A, T, C, or G 17 tttttttttt tttttggtta caaaagtatt
tattttataa aacttgtatt taaaatagag 60 cttatctgtc tactcacaaa
tcctaattta aaacataaca cattatcctt agctaatctg 120 atgttaacct
ttacaatcaa cactcatttt tgtaatttta ttaagaacct gtactaaatg 180
aagtttttaa tcagaaaaca ttccctttta tcttaaaagt gcttcttaaa tgaaggcacc
240 aacaagaact actttcagat ggtacagaat ttcttatttc ttgaagactc
tgtggttgac 300 cacttcttca ttagttacct gcagcaagac accttcctgc
caaaggaaaa aaaaaagtat 360 ctgaagaagt ttatcatgtt tgtccaaaga
acctaagtaa cttcagtggt ggttttagga 420 ttaaagcaga ctcactgatg
tgtatacgcc ctgaatatca catttctgga aaggcagtaa 480 agcctagaaa
tcagaaggcg ggcggtttta aagaaatttc aatagccaac ctacaacant 540
ttagggcaaa gataatgggc aaaaant 567 18 346 DNA Eukaryote misc_feature
(1)...(346) r = A or G y = T or C m = A or C k = G or T s = G or C
w = A or T b = G, C, or T; not A d = A, G, or T; not C h = A, C, or
T; not G v = A, G, or C; not T n = A, T, G, or C 18 acgatatmta
ywgarrtwya wctstthact gaatmwhatg cacaaatatt aactagtrrt 60
ttattaaaca gatatsattt agaacaagac ttaawkaaat acaaatcctt aggtacgrtt
120 taatatcatg ttcadgatgt ttgaagagtt taaaaagaat cactgattaa
gkkaagcatc 180 cbcacttttc tttgagaabc caaacctttt aggnaaadac
cccattccaa attttgtccc 240 chatttcagr cckkcagaaa gtctctaaca
tsaagagtcc tcaacggggn gtaactcava 300 wctcctatca agtgcagtaa
cctagctctc ccggdggcca tggcgt 346 19 803 DNA Eukaryote misc_feature
(1)...(803) n = A, C, T, or G 19 aaactaaaca gtgttttgtt aattcttctg
cattcggact attgcaggca ttagagcatc 60 cagagctacg aagggctggc
tgcagcagca ccgccctttg taagccagca gaccagcctt 120 aactgtgggc
ttgactcctg tgagctggcc tcagtgtgac tcagaaatgt ttgattagca 180
gatgagagag cgaggacaca ccacgagggc tgcgttctct tcctccagcg ctgtgcagga
240 cagtttcttc tcaccctagc ctttttaaat gcaccagaag tacagacagt
tgcactacac 300 aaaccctttg aacacttgta gaaatcagtc caccgtagat
tagacagaat caccttccaa 360 tcctttgact tcttttcctt tcatttgaac
aattgtataa taattgatta ttgtcaaatt 420 tttgtctgtg gtagtatcgc
tttaatttat cttagtacat caacgttttg atttaaaaaa 480 gaattaaaac
aacaaaaaaa gtcacttaga agccatgaac tttttttttt ngatngggaa 540
attttcttgt ttngaaaatt atcattgggg ttcctccgga aancttgtaa gattggntta
600 taaggtacct gggangttca naacnggtgg ntataccctt ttttaaggga
aattaatgat 660 ttngagtttt tgggccaact ncgggantgg cagggaaacc
anncnggggn ggggtttaaa 720 ttntgtgagg gttttttggg cctnaatttt
ttgcataatt ttcacctngn aacctttnaa 780 nnctnggaaa aaaaaaaaaa cnt 803
20 2540 DNA Eukaryote CDS (3)...(2348) misc_feature (1)...(2540) n
= A,T,C or G 20
tg cag ccg ccc ttg gaa ctg cat gtc agg aag cat ccc ttt gtg tat 47
Gln Pro Pro Leu Glu Leu His Val Arg Lys His Pro Phe Val Tyr 1 5 10
15 gtc tgt gct ata tgt ctc aag aaa ttt gtc agc tca atc agg ctg cgc
95 Val Cys Ala Ile Cys Leu Lys Lys Phe Val Ser Ser Ile Arg Leu Arg
20 25 30 tcc cat atc cga gag gtg cat ggg gcg gcc cag gag acc ttg
gtt ttt 143 Ser His Ile Arg Glu Val His Gly Ala Ala Gln Glu Thr Leu
Val Phe 35 40 45 act agc tcc atc aac cag agt ttc tgc ctc ctg gag
cct ggt ggg gat 191 Thr Ser Ser Ile Asn Gln Ser Phe Cys Leu Leu Glu
Pro Gly Gly Asp 50 55 60 atc cag cag gaa gcc ttg gga aac cag cta
tca ctg aca gct gag gaa 239 Ile Gln Gln Glu Ala Leu Gly Asn Gln Leu
Ser Leu Thr Ala Glu Glu 65 70 75 ttt gtg tgt cca gaa att gat gta
cgt aag ggg gag gtt tgt cct ggg 287 Phe Val Cys Pro Glu Ile Asp Val
Arg Lys Gly Glu Val Cys Pro Gly 80 85 90 95 gaa gct cag cct gag gtg
ggg ctg agg gag ttg gag gcc cct gga gaa 335 Glu Ala Gln Pro Glu Val
Gly Leu Arg Glu Leu Glu Ala Pro Gly Glu 100 105 110 gca tgt gcc cca
gcc gtg ccc ttg gcc aac ccc cag agt gtc agt gtt 383 Ala Cys Ala Pro
Ala Val Pro Leu Ala Asn Pro Gln Ser Val Ser Val 115 120 125 tcc ctg
tcc ccc tgc aaa ctg gaa acc act gtg gtc aat tcc gac ctc 431 Ser Leu
Ser Pro Cys Lys Leu Glu Thr Thr Val Val Asn Ser Asp Leu 130 135 140
aac tct ctt gga gtg gtt tca gat gat ttt tta ctg aaa act gat acc 479
Asn Ser Leu Gly Val Val Ser Asp Asp Phe Leu Leu Lys Thr Asp Thr 145
150 155 tct tct gct gag cct cat gct gct gct gag cta acc tca gac aca
cag 527 Ser Ser Ala Glu Pro His Ala Ala Ala Glu Leu Thr Ser Asp Thr
Gln 160 165 170 175 cat cga ggc tca gcc cag act cag ggt gaa gaa gtc
aca ctg ctg ctg 575 His Arg Gly Ser Ala Gln Thr Gln Gly Glu Glu Val
Thr Leu Leu Leu 180 185 190 gcc aag gcc aaa agt act gga cca gac tca
gag agt cct cca agt gga 623 Ala Lys Ala Lys Ser Thr Gly Pro Asp Ser
Glu Ser Pro Pro Ser Gly 195 200 205 ggg cag aat gtg ggt gct ctg cca
gcc agt gaa tct gac tct aac agg 671 Gly Gln Asn Val Gly Ala Leu Pro
Ala Ser Glu Ser Asp Ser Asn Arg 210 215 220 tgt ctc agg gca aac cca
gca gag acc tca gac ctc ctt cct aca gtg 719 Cys Leu Arg Ala Asn Pro
Ala Glu Thr Ser Asp Leu Leu Pro Thr Val 225 230 235 gct gat gga gga
gac ctc ggt gtg tgc cag cct gac tct tgc acg tcg 767 Ala Asp Gly Gly
Asp Leu Gly Val Cys Gln Pro Asp Ser Cys Thr Ser 240 245 250 255 tcc
tct gag cac cac cct ggc agc aca gca ttc atg aag gtc cta gac 815 Ser
Ser Glu His His Pro Gly Ser Thr Ala Phe Met Lys Val Leu Asp 260 265
270 agt ctc cag aag aag cag atg aac acc agt ctt tgc gag cgg atc cgg
863 Ser Leu Gln Lys Lys Gln Met Asn Thr Ser Leu Cys Glu Arg Ile Arg
275 280 285 aag gtt tat gga gac ctg gag tgt gaa tac tgt ggc aaa ctt
ttt tgg 911 Lys Val Tyr Gly Asp Leu Glu Cys Glu Tyr Cys Gly Lys Leu
Phe Trp 290 295 300 tac caa gtg cat ttt gac atg cat gtc cgc acc cac
acc cgg gaa cat 959 Tyr Gln Val His Phe Asp Met His Val Arg Thr His
Thr Arg Glu His 305 310 315 ctg tat tat tgc tcc cag tgt cac tac tct
tcc atc acc aaa aac tgc 1007 Leu Tyr Tyr Cys Ser Gln Cys His Tyr
Ser Ser Ile Thr Lys Asn Cys 320 325 330 335 ctt aaa cgc cat gta att
cag aaa cac agt aac atc ttg ctg aag tgt 1055 Leu Lys Arg His Val
Ile Gln Lys His Ser Asn Ile Leu Leu Lys Cys 340 345 350 ccc act gac
ggc tgt gac tac tcg act cca gat aaa tat aag cta cag 1103 Pro Thr
Asp Gly Cys Asp Tyr Ser Thr Pro Asp Lys Tyr Lys Leu Gln 355 360 365
gcc cac ctt aaa gtt cac aca gag ctg gac aaa agg agt tat tct tgt
1151 Ala His Leu Lys Val His Thr Glu Leu Asp Lys Arg Ser Tyr Ser
Cys 370 375 380 cct gta tgt gaa aaa tct ttt tca gaa gac cga ttg ata
aag tca cat 1199 Pro Val Cys Glu Lys Ser Phe Ser Glu Asp Arg Leu
Ile Lys Ser His 385 390 395 atc aag act aat cat cca gag gtc tcc atg
aat acc att tct gag gtt 1247 Ile Lys Thr Asn His Pro Glu Val Ser
Met Asn Thr Ile Ser Glu Val 400 405 410 415 ctt ggg aga aga gtc cag
ctc aaa ggg cta att gga aag cga gcc atg 1295 Leu Gly Arg Arg Val
Gln Leu Lys Gly Leu Ile Gly Lys Arg Ala Met 420 425 430 aag tgt ccg
tat tgc gat ttc tat ttc atg aag aat ggc tca gac ctt 1343 Lys Cys
Pro Tyr Cys Asp Phe Tyr Phe Met Lys Asn Gly Ser Asp Leu 435 440 445
cag cgg cac atc tcn gct cac gag ggt gtg aag ccc ttc aaa tgt tct
1391 Gln Arg His Ile Ser Ala His Glu Gly Val Lys Pro Phe Lys Cys
Ser 450 455 460 ttg tgt gag tat gca act cgt agc aag agc aac ctc aaa
gct cat atg 1439 Leu Cys Glu Tyr Ala Thr Arg Ser Lys Ser Asn Leu
Lys Ala His Met 465 470 475 aat cgt cac agc act gag aag act cac ctc
tgt gac atg tgt ggc aag 1487 Asn Arg His Ser Thr Glu Lys Thr His
Leu Cys Asp Met Cys Gly Lys 480 485 490 495 aaa ttc aaa tcc aaa ggg
aca tta aag agt cat aag ctc ctt cac aca 1535 Lys Phe Lys Ser Lys
Gly Thr Leu Lys Ser His Lys Leu Leu His Thr 500 505 510 tct gat ggg
aag caa ttc aag tgc acg gtg tgt gac tac aca gct gcc 1583 Ser Asp
Gly Lys Gln Phe Lys Cys Thr Val Cys Asp Tyr Thr Ala Ala 515 520 525
cag aaa cca cag ctg ctg cga cac atg gag cag gat gcc tcc ttc aag
1631 Gln Lys Pro Gln Leu Leu Arg His Met Glu Gln Asp Ala Ser Phe
Lys 530 535 540 cct ttc cgc tgc gct cac tgt cat tat tca tgt aac atc
tct gga tct 1679 Pro Phe Arg Cys Ala His Cys His Tyr Ser Cys Asn
Ile Ser Gly Ser 545 550 555 ctg aaa cgg cac tac aac agg aag cac ccc
aac gag gag tat gcc aac 1727 Leu Lys Arg His Tyr Asn Arg Lys His
Pro Asn Glu Glu Tyr Ala Asn 560 565 570 575 gtg ggc agc ggg gag ctt
gca gct gaa gcc ctc atc caa caa ggt ggt 1775 Val Gly Ser Gly Glu
Leu Ala Ala Glu Ala Leu Ile Gln Gln Gly Gly 580 585 590 ctg aag tgt
cct gtt tgc agc ttt gtg tat gga acc aaa tgg gag ttc 1823 Leu Lys
Cys Pro Val Cys Ser Phe Val Tyr Gly Thr Lys Trp Glu Phe 595 600 605
aac aga cac ttg aag aac aag cat ggc ttg aag cca gcg aca gag act
1871 Asn Arg His Leu Lys Asn Lys His Gly Leu Lys Pro Ala Thr Glu
Thr 610 615 620 ccc gag gag ccc tcc acc cag tat ctc tac atc acc gag
gct gaa gat 1919 Pro Glu Glu Pro Ser Thr Gln Tyr Leu Tyr Ile Thr
Glu Ala Glu Asp 625 630 635 gtt cag ggg aca caa gca gct gta gct gca
ctt cag gac ctg cga tat 1967 Val Gln Gly Thr Gln Ala Ala Val Ala
Ala Leu Gln Asp Leu Arg Tyr 640 645 650 655 acc tcc gag agt ggt gat
cga ctt gac ccc aca gct gtg aat atc ctg 2015 Thr Ser Glu Ser Gly
Asp Arg Leu Asp Pro Thr Ala Val Asn Ile Leu 660 665 670 cag cag atc
att gaa ctg ggt tca gag act cac gat gct gct gcc gtg 2063 Gln Gln
Ile Ile Glu Leu Gly Ser Glu Thr His Asp Ala Ala Ala Val 675 680 685
gcc tcc gtg gtt gcc atg gcg cct ggg aca gtg act gtt gta aag cag
2111 Ala Ser Val Val Ala Met Ala Pro Gly Thr Val Thr Val Val Lys
Gln 690 695 700 gtc acc gat gag gaa ccc aat tcc aac cat aca gtc atg
atc cag gag 2159 Val Thr Asp Glu Glu Pro Asn Ser Asn His Thr Val
Met Ile Gln Glu 705 710 715 act ctg cag cag gcc tct gtg gag ttg gcc
gag cag cac cat ctg gtg 2207 Thr Leu Gln Gln Ala Ser Val Glu Leu
Ala Glu Gln His His Leu Val 720 725 730 735 gtg tcc tct gat gac gtg
gag ggc att gag aca gtg aca gtg tac aca 2255 Val Ser Ser Asp Asp
Val Glu Gly Ile Glu Thr Val Thr Val Tyr Thr 740 745 750 cag ggt ggg
gag gcc tca gag ttc atc gtg tac gtg caa gag gct gtc 2303 Gln Gly
Gly Glu Ala Ser Glu Phe Ile Val Tyr Val Gln Glu Ala Val 755 760 765
cag ccc atg gag gag cag gtc ggg gag cag cca gcc aca gaa ctc 2348
Gln Pro Met Glu Glu Gln Val Gly Glu Gln Pro Ala Thr Glu Leu 770 775
780 tagagaatcc ctgcctcctt tggcagccag cctttgtggg cctgaagacc
tcctaaccca 2408 ccaggtccat ccctggctct tcttgcccac tggccccaga
taaatttctc cataactgtc 2468 ctctgtgtgg tcaaagccag gagagtatca
tgaagagaga gagagagaga gactagtctc 2528 cgagtttttt tt 2540 21 782 PRT
Eukaryote 21 Gln Pro Pro Leu Glu Leu His Val Arg Lys His Pro Phe
Val Tyr Val 1 5 10 15 Cys Ala Ile Cys Leu Lys Lys Phe Val Ser Ser
Ile Arg Leu Arg Ser 20 25 30 His Ile Arg Glu Val His Gly Ala Ala
Gln Glu Thr Leu Val Phe Thr 35 40 45 Ser Ser Ile Asn Gln Ser Phe
Cys Leu Leu Glu Pro Gly Gly Asp Ile 50 55 60 Gln Gln Glu Ala Leu
Gly Asn Gln Leu Ser Leu Thr Ala Glu Glu Phe 65 70 75 80 Val Cys Pro
Glu Ile Asp Val Arg Lys Gly Glu Val Cys Pro Gly Glu 85 90 95 Ala
Gln Pro Glu Val Gly Leu Arg Glu Leu Glu Ala Pro Gly Glu Ala 100 105
110 Cys Ala Pro Ala Val Pro Leu Ala Asn Pro Gln Ser Val Ser Val Ser
115 120 125 Leu Ser Pro Cys Lys Leu Glu Thr Thr Val Val Asn Ser Asp
Leu Asn 130 135 140 Ser Leu Gly Val Val Ser Asp Asp Phe Leu Leu Lys
Thr Asp Thr Ser 145 150 155 160 Ser Ala Glu Pro His Ala Ala Ala Glu
Leu Thr Ser Asp Thr Gln His 165 170 175 Arg Gly Ser Ala Gln Thr Gln
Gly Glu Glu Val Thr Leu Leu Leu Ala 180 185 190 Lys Ala Lys Ser Thr
Gly Pro Asp Ser Glu Ser Pro Pro Ser Gly Gly 195 200 205 Gln Asn Val
Gly Ala Leu Pro Ala Ser Glu Ser Asp Ser Asn Arg Cys 210 215 220 Leu
Arg Ala Asn Pro Ala Glu Thr Ser Asp Leu Leu Pro Thr Val Ala 225 230
235 240 Asp Gly Gly Asp Leu Gly Val Cys Gln Pro Asp Ser Cys Thr Ser
Ser 245 250 255 Ser Glu His His Pro Gly Ser Thr Ala Phe Met Lys Val
Leu Asp Ser 260 265 270 Leu Gln Lys Lys Gln Met Asn Thr Ser Leu Cys
Glu Arg Ile Arg Lys 275 280 285 Val Tyr Gly Asp Leu Glu Cys Glu Tyr
Cys Gly Lys Leu Phe Trp Tyr 290 295 300 Gln Val His Phe Asp Met His
Val Arg Thr His Thr Arg Glu His Leu 305 310 315 320 Tyr Tyr Cys Ser
Gln Cys His Tyr Ser Ser Ile Thr Lys Asn Cys Leu 325 330 335 Lys Arg
His Val Ile Gln Lys His Ser Asn Ile Leu Leu Lys Cys Pro 340 345 350
Thr Asp Gly Cys Asp Tyr Ser Thr Pro Asp Lys Tyr Lys Leu Gln Ala 355
360 365 His Leu Lys Val His Thr Glu Leu Asp Lys Arg Ser Tyr Ser Cys
Pro 370 375 380 Val Cys Glu Lys Ser Phe Ser Glu Asp Arg Leu Ile Lys
Ser His Ile 385 390 395 400 Lys Thr Asn His Pro Glu Val Ser Met Asn
Thr Ile Ser Glu Val Leu 405 410 415 Gly Arg Arg Val Gln Leu Lys Gly
Leu Ile Gly Lys Arg Ala Met Lys 420 425 430 Cys Pro Tyr Cys Asp Phe
Tyr Phe Met Lys Asn Gly Ser Asp Leu Gln 435 440 445 Arg His Ile Ser
Ala His Glu Gly Val Lys Pro Phe Lys Cys Ser Leu 450 455 460 Cys Glu
Tyr Ala Thr Arg Ser Lys Ser Asn Leu Lys Ala His Met Asn 465 470 475
480 Arg His Ser Thr Glu Lys Thr His Leu Cys Asp Met Cys Gly Lys Lys
485 490 495 Phe Lys Ser Lys Gly Thr Leu Lys Ser His Lys Leu Leu His
Thr Ser 500 505 510 Asp Gly Lys Gln Phe Lys Cys Thr Val Cys Asp Tyr
Thr Ala Ala Gln 515 520 525 Lys Pro Gln Leu Leu Arg His Met Glu Gln
Asp Ala Ser Phe Lys Pro 530 535 540 Phe Arg Cys Ala His Cys His Tyr
Ser Cys Asn Ile Ser Gly Ser Leu 545 550 555 560 Lys Arg His Tyr Asn
Arg Lys His Pro Asn Glu Glu Tyr Ala Asn Val 565 570 575 Gly Ser Gly
Glu Leu Ala Ala Glu Ala Leu Ile Gln Gln Gly Gly Leu 580 585 590 Lys
Cys Pro Val Cys Ser Phe Val Tyr Gly Thr Lys Trp Glu Phe Asn 595 600
605 Arg His Leu Lys Asn Lys His Gly Leu Lys Pro Ala Thr Glu Thr Pro
610 615 620 Glu Glu Pro Ser Thr Gln Tyr Leu Tyr Ile Thr Glu Ala Glu
Asp Val 625 630 635 640 Gln Gly Thr Gln Ala Ala Val Ala Ala Leu Gln
Asp Leu Arg Tyr Thr 645 650 655 Ser Glu Ser Gly Asp Arg Leu Asp Pro
Thr Ala Val Asn Ile Leu Gln 660 665 670 Gln Ile Ile Glu Leu Gly Ser
Glu Thr His Asp Ala Ala Ala Val Ala 675 680 685 Ser Val Val Ala Met
Ala Pro Gly Thr Val Thr Val Val Lys Gln Val 690 695 700 Thr Asp Glu
Glu Pro Asn Ser Asn His Thr Val Met Ile Gln Glu Thr 705 710 715 720
Leu Gln Gln Ala Ser Val Glu Leu Ala Glu Gln His His Leu Val Val 725
730 735 Ser Ser Asp Asp Val Glu Gly Ile Glu Thr Val Thr Val Tyr Thr
Gln 740 745 750 Gly Gly Glu Ala Ser Glu Phe Ile Val Tyr Val Gln Glu
Ala Val Gln 755 760 765 Pro Met Glu Glu Gln Val Gly Glu Gln Pro Ala
Thr Glu Leu 770 775 780 22 1012 DNA Eukaryote misc_feature
(1)...(1012) r = G or A y = C or T m = A or C k = G or T s = G or C
w = A or T 22 tggatctact tgttaatggt ttcatggaag caatcagcaa
tatgtgatat gaactgctgc 60 attacttatt atactcgtgg aactgagata
tttarmsrsm gcttwwyttt tttttttytt 120 agtgtaaaat acttaagcgt
ttccactatt ggaagaaaag catatatggg tattttgtat 180 tgtaacttgt
ttaaaaggac agtctttttt aaycttccca cttaaatgct tttaaaatat 240
gtaatacaat ttgaagcttg tttaaaaata gaattaaatg tcttawatag kgctackgtt
300 ttggaattag aaagtgatca aatacaaaac attttaaaat taagcccaga
aaacaaaata 360 gtgtttaaag ttagtttagt ataaaagaaa tttataagat
tttttcttca atataagata 420 cctcacttga aaataaagaa agcacagcac
attaaagtaa ttctcatgag aacaccccat 480 tagaataatt gctaaatcta
ggacaccttt tgagttgtga gtttgtgata catgtagtca 540 ccattagctt
ttctgctgga aggacttccc gtagtaattt taaggcagtg taatagttca 600
attaccccac agtttctaac ctgggaaggc agtatgtgaa tggtcccttc tgcaactacg
660 gaaacacatt agctacattg agcataactc gattgataat tttgccagtg
catatagttt 720 tatgattaaa attgctgtgg ttggttgcat tacacgacac
acaaaactgt cctctacctc 780 acatgaaata aatattttat atggttttac
taaaaaaatg actcatctat ctggttactt 840 agtttacaaa ttttggatta
tatttattga aacatgacat actgtgctct tagcttatac 900 ctcaatcgta
ttttgtgctg tttgccattt tcatgccttg tatataactt gtatagattg 960
gatgatattc ccaataaaca cttttaatkc caawraaaaa aaaaaaaaaa aa 1012 23
1747 DNA Eukaryote misc_feature (1)...(1747) n = A,T,C or G 23
taatgtttat gatacaaagc tactcactct ggagccttct cattacagaa tctcttgact
60 tttatacacc cagcctgttg ttactttgtt caggttgcag aatgagtttc
ctctggtttc 120 ctcctagagg agttttcctg atgaaatgct agtagcacct
ccccgacata cagcgggtgg 180 gtggggcaca ctttgctgtg ctctgatggt
acacacaaga agcagttgta atttgtcttt 240 ctgtttaaga gtgaccatag
ctagatatgt gtgtgtgact tcagaaaatt aaaatgcttt 300 ccgaactttt
cctgttaata gaggtgtgaa gtactcattc atgtgcatga ggaaagtgga 360
ttccacggac gcacaccgct tcctatgtaa ctcacaatgc tctgtacagt ttttatatgt
420 agtcttacaa aggtcttatg aaatttatat aatggatttt ttcttttaaa
ttataaaata 480 ctaaatatct taaagattgt tttggacttt tgtatgttta
aatgttatct taaaacttgc 540 acaaatggac catgatgact ctttgatctt
aaaatcagga atttacagtc agctaagaaa 600 aatgtggata ggttaataat
ccacagtggg agtatctgct aggagcagga attgtagatg 660 acatgaattc
cgtgatttga ggaagggcag cctctgcact tttctttgtt tttgtttttt 720
gcacatgaag tctgacattt ttaccatcga atttcacatt actagatggt tggcttggga
780 tttacctagg ggaaattctt agcaactttg tactttgttg tttttgttct
gtttggtctc 840 cagcttgcag agaccctctt gcctctgtct cccaagtgtt
gggttggcag gatgagcccc 900 accaccgctg gccctgtgca gttcttttgg
gatgtccctg aaagcagctg tggcattatc 960 ttctgtttca tgtgtcccga
gctgtctcat ggtactacat gcagtgacct gagatctgcg 1020 ttaaggaata
acttaggaga aaacggctgt cactgtcctc cccgctgtga gacaccagag 1080
ttatcacacc tgttatggtc atactttgtg ttatgatact gatgtctaag gcaatttttc
1140 tactttccaa aagggagttt gtttcctaaa tatattgtga cctaaatgtg
gttttattct 1200 gctatgttct ataatttatg tattgacttt tgtaacctcc
ttgggagaaa catgttaagt 1260 ggcacaggga ccatatatgt cattttattt
agctctggag aaggaaacca caggcgtttg 1320 taaaatagca ttagcttaga
tgtcagttca ttgtgcttgg ctgtgtggga ggcagactca 1380 aggacttgca
ccatttattt ttctgacaga agtgttctgc ttatgtgctg cttagtaagt 1440
gtgatttttc tagtcttgat gaaacttgcc tcgtgacatt gttgagcgta gtcttcactt
1500 tccagaagat gaaatgatgt gccatcattt tctgtctaaa cttcctttaa
agtaattttt 1560 aatcagctgt aaatatcata tctcctactg ttgaaagtaa
ctttaattta cattgcacca 1620 tatagcttga aaaccaactt tgaaattctg
tactcctcca caagtgacct ccgctaaaat 1680 acccatagga agcttacttt
gtgcatgcnt gctttgtgtg ccggttgccg tcctaanggt 1740 tgctttg 1747 24
571 DNA Eukaryote misc_feature (1)...(571) n = A,T,C or G 24
tttttttttt ttttttttag tgtaaaatac ttaagcgttt ccactattgg aagaaaagca
60 tatatgggta ttttgtattg taacttgttt aaaaggacag tcttttttaa
tcttcccact 120 taaatgcttt taaaatatgt aatacaattt gaagcttgtt
taaaaataga attaaatgtc 180 ttatatagtg ctactgtttt ggaattagaa
agtgatcaaa tacaaaacat tttaaaatta 240 agcccagaaa acaaaatagt
gtttaaagtt agtttagtat aaaagaaatt tatgagattt 300 tttcttcaat
ataagatacc tcacttgaaa ataaagaaag cacagcacat taaagtaatt 360
ctcatgagaa caccccatta gaataattgc taaatctagg acaccttttg agttgtgaag
420 tttgtgatac atgtagtcac cattagcttt tctgctggaa ggacttcccg
tagtaatttt 480 aaagnagtgt aataagttca attancccac aagtttctaa
nctgggaaag naantatggt 540 gaatggnccc ttctgcaact acgggaacac a 571 25
619 DNA Eukaryote 25 tttttttttt tttttttttt tggcattaaa agtgtttatt
gggaatatca tccaatctat 60 acaagttata tacaaggcat gaaaatggca
aacagcacaa aatacgattg aggtataagc 120 taagagcaca gtatgtcatg
tttcaataaa tataatccaa aatttgtaaa ctaagtaacc 180 agatagatga
gtcatttttt tagtaaaacc atataaaata tttatttcat gtgaggtaga 240
ggacagtttt gtgtgtcgtg taatgcaacc aaccacagca attttaatca taaaactata
300 tgcactggca aaattatcaa tcgagttatg ctcaatgtag ctaatgtgtt
tccgtagttg 360 cagaagggac cattcacata ctgccttccc aggttagaaa
ctgtggggta attgaactat 420 tacactgcct taaaattact acgggaagtc
cttccagcag aaaagctaat ggtgactaca 480 tgtatcacaa actcacaact
caaaaggtgt cctagattta gcaattattc taatggggtg 540 ttctcatgag
aattacttta atgtgctgtg ctttctttat ttcaagtgag gtatcttata 600
ttgaagaaaa aatccataa 619 26 2995 DNA Eukaryote CDS (218)...(1960)
26 tgcggccgcc ggggccgggg ctgagccagt ctctcccgcc gccgccggac
gcgcagacct 60 gggcaggctg caccgacggc cgcctggccg agcgcactgc
aggtcgctgc gcgcgctgcg 120 accccggggc ccggacgcga gtggctgcgg
tgtcctgggc gagcactgct agtttaggcc 180 gtctgtcctc agctgcttgg
aacccctaca tcccacc atg gct ggg ata cag aag 235 Met Ala Gly Ile Gln
Lys 1 5 agg aag ttt gac cag ctg gaa gag gac gac tgc agc tcc tcc tcc
ttg 283 Arg Lys Phe Asp Gln Leu Glu Glu Asp Asp Cys Ser Ser Ser Ser
Leu 10 15 20 tcc tct ggc gat ctc tct ccc tct cct ccc agc tct tct
gcc tcc cct 331 Ser Ser Gly Asp Leu Ser Pro Ser Pro Pro Ser Ser Ser
Ala Ser Pro 25 30 35 gcc tgg acc tct gag gag gag gga ctg ggt gat
cag cca ccc cag cct 379 Ala Trp Thr Ser Glu Glu Glu Gly Leu Gly Asp
Gln Pro Pro Gln Pro 40 45 50 gat cag gac tcc agt ggc atc cag agt
tta acg ccc cca tcc atc ctg 427 Asp Gln Asp Ser Ser Gly Ile Gln Ser
Leu Thr Pro Pro Ser Ile Leu 55 60 65 70 aag cgg gct cct cgg gag cgt
ccg ggt cac gtg gcc ttc gat ggc atc 475 Lys Arg Ala Pro Arg Glu Arg
Pro Gly His Val Ala Phe Asp Gly Ile 75 80 85 act gtc tac tat ttc
ccg cgg tgc cag gga ttc acc agt gtg ccc agc 523 Thr Val Tyr Tyr Phe
Pro Arg Cys Gln Gly Phe Thr Ser Val Pro Ser 90 95 100 cat ggt ggc
tgt acc ctg ggc atg gct tct cgt cat agc acc tgc cgc 571 His Gly Gly
Cys Thr Leu Gly Met Ala Ser Arg His Ser Thr Cys Arg 105 110 115 ctc
ttc tcc tta gcc gag ttt aaa cag gag cag ttc cgg gct cgg cgt 619 Leu
Phe Ser Leu Ala Glu Phe Lys Gln Glu Gln Phe Arg Ala Arg Arg 120 125
130 gag aag ctc cgt cgg cgt tta aag gag gag aag cta gag atg ctg aaa
667 Glu Lys Leu Arg Arg Arg Leu Lys Glu Glu Lys Leu Glu Met Leu Lys
135 140 145 150 tgg aag ctt tca gtg tcc gga gtt ccg gag gca ggg gca
gac gtg ccg 715 Trp Lys Leu Ser Val Ser Gly Val Pro Glu Ala Gly Ala
Asp Val Pro 155 160 165 ctc aca gtg gac gcc atc gat gac gct tct gta
gag gag gac ttg gca 763 Leu Thr Val Asp Ala Ile Asp Asp Ala Ser Val
Glu Glu Asp Leu Ala 170 175 180 gtg gcc gtg gca ggt ggc cgc ctg gag
gaa gcg aat ttc cta cag ccc 811 Val Ala Val Ala Gly Gly Arg Leu Glu
Glu Ala Asn Phe Leu Gln Pro 185 190 195 tat cca cct cgg cag cga cgg
gcc cta ctt cgc gct tcc ggt gtt cga 859 Tyr Pro Pro Arg Gln Arg Arg
Ala Leu Leu Arg Ala Ser Gly Val Arg 200 205 210 agg att gac cga gag
gag aag cac gag ctg cag gcg cta cgc caa tcc 907 Arg Ile Asp Arg Glu
Glu Lys His Glu Leu Gln Ala Leu Arg Gln Ser 215 220 225 230 cgg gag
gat tgt ggt tgt cac tgt gat ggc gtc tgt gac cct gag acc 955 Arg Glu
Asp Cys Gly Cys His Cys Asp Gly Val Cys Asp Pro Glu Thr 235 240 245
tgc agt tgc atc ctg gcg ggc att aaa tgc cag atg gat cac acg tcc
1003 Cys Ser Cys Ile Leu Ala Gly Ile Lys Cys Gln Met Asp His Thr
Ser 250 255 260 ttc ccc tgt ggc tgc tgc agc gag ggc tgt gag aac ccc
cat ggt cga 1051 Phe Pro Cys Gly Cys Cys Ser Glu Gly Cys Glu Asn
Pro His Gly Arg 265 270 275 gtg gaa ttc aat cag gcg aga gtt cag aca
cac ttc atc cac acg ctc 1099 Val Glu Phe Asn Gln Ala Arg Val Gln
Thr His Phe Ile His Thr Leu 280 285 290 acc cgc ctg cag atg gag cag
ggt gcg gag agt ttg ggg gac ccg gag 1147 Thr Arg Leu Gln Met Glu
Gln Gly Ala Glu Ser Leu Gly Asp Pro Glu 295 300 305 310 tcc ccc atg
gag gac gtt cct gtc gaa caa acc gtg gtt tcc ccc ttt 1195 Ser Pro
Met Glu Asp Val Pro Val Glu Gln Thr Val Val Ser Pro Phe 315 320 325
cct cct tcc aaa ccc act atg agc aat gac ctg ggg gac agc agc tgt
1243 Pro Pro Ser Lys Pro Thr Met Ser Asn Asp Leu Gly Asp Ser Ser
Cys 330 335 340 ggc agc gac atg aca gac tct tcc acg acc tac tcc tct
ggc ggc agt 1291 Gly Ser Asp Met Thr Asp Ser Ser Thr Thr Tyr Ser
Ser Gly Gly Ser 345 350 355 ggc agc cgc agc gag gct ccg aac cat ctt
gcc cac ccc agc ctg cca 1339 Gly Ser Arg Ser Glu Ala Pro Asn His
Leu Ala His Pro Ser Leu Pro 360 365 370 ggt tcc agc ttc cgg tct ggc
ata gat gaa gac agc ctg gaa cag atc 1387 Gly Ser Ser Phe Arg Ser
Gly Ile Asp Glu Asp Ser Leu Glu Gln Ile 375 380 385 390 ctg aat ttc
agt gac tct gac ctc ggt att gag gaa gaa gag gag gag 1435 Leu Asn
Phe Ser Asp Ser Asp Leu Gly Ile Glu Glu Glu Glu Glu Glu 395 400 405
gga ggg agt gtg ggc aac ttg gat aac ctc agc tgt ttt cat ttg gct
1483 Gly Gly Ser Val Gly Asn Leu Asp Asn Leu Ser Cys Phe His Leu
Ala 410 415 420 gac atc ttt ggt acc ggt gac ccc ggc agc ctg gct agc
tgg aca cac 1531 Asp Ile Phe Gly Thr Gly Asp Pro Gly Ser Leu Ala
Ser Trp Thr His 425 430 435 agc cag ttt ggc tct agc ctt cca tcg ggc
atc cta gat gag aat gcc 1579 Ser Gln Phe Gly Ser Ser Leu Pro Ser
Gly Ile Leu Asp Glu Asn Ala 440 445 450 aac ctg gac gcc agc tgc ttc
cta agc agc gga ctc gaa ggg ttg aga 1627 Asn Leu Asp Ala Ser Cys
Phe Leu Ser Ser Gly Leu Glu Gly Leu Arg 455 460 465 470 gaa ggt agc
ctc ccc agc agt tct ggg tcc cct gag ggg gaa gcc gcc 1675 Glu Gly
Ser Leu Pro Ser Ser Ser Gly Ser Pro Glu Gly Glu Ala Ala 475 480 485
cag agc agc tcc ttg gac ctc agt tta tcc tcc tgt gac tcc ttt gag
1723 Gln Ser Ser Ser Leu Asp Leu Ser Leu Ser Ser Cys Asp Ser Phe
Glu 490 495 500 ctt ctc caa tct ctg cca gat tat agt ctg ggg cct cac
tat act tcc 1771 Leu Leu Gln Ser Leu Pro Asp Tyr Ser Leu Gly Pro
His Tyr Thr Ser 505 510 515 cga agg gta tct ggc agc ctg gac agc ctt
gag acc ttc cac cct tcg 1819 Arg Arg Val Ser Gly Ser Leu Asp Ser
Leu Glu Thr Phe His Pro Ser 520 525 530 ccc agc ttc tct cca ccg agg
gat gcc agc ttc ctg gat tct ctc ata 1867 Pro Ser Phe Ser Pro Pro
Arg Asp Ala Ser Phe Leu Asp Ser Leu Ile 535 540 545 550 ggc ctg tct
gag ccg gtt aca gat gtc ctg gcg ccc ctt ctg gag agc 1915 Gly Leu
Ser Glu Pro Val Thr Asp Val Leu Ala Pro Leu Leu Glu Ser 555 560 565
cag ttt gag gac act gct gtg gtg cct ttg gac cct gtg cct gtg 1960
Gln Phe Glu Asp Thr Ala Val Val Pro Leu Asp Pro Val Pro Val 570 575
580 taaggattga gatgactttt tcctgccctg agaccctgtt gctgcttttt
atgtgatctt 2020 ggtgtccccc aaggtctgtg tatgtaacgg tctcccgtgg
gctggttctg cccccgtgcc 2080 atgtgggcaa tcctctattt ttacagtaac
actctagatt tatttatttt tttatgtttt 2140 tctgtactga agggagggtg
ggaagggtat ccctctttca atgcctggcc tctatgtcca 2200 aacagaggtc
tcccacctcc tactgtatgc ctggaggagg aaggggcggg gttcacatcc 2260
cctctttctg tactgtaaaa tgctccttgg tccaaagaca gctgaaaagc aggccttagg
2320 gtttcctgtg gaccgtggga gctaggtctt ctggactctg aagatgtaat
ttatttctgt 2380 aatttatttg gggactgaga cagcagtggt tgggcctctc
tggcaggtgg gcggtgttga 2440 ggcaaagtct tcggtgtccc ccgccggtct
gggcttcggt gtggcgtgta ggttcgagct 2500 gagcagacgg aggctgtgct
tgaccatcgg tgatcaaaac tccctctgcc ccctgcccag 2560 acgctctaac
atgccctctg tccatttccc tctccccaag gccatgggtt ataaaggccc 2620
tatgtaggat ggggagccag aggccctaag acatgaagca caccccagat cactgtctct
2680 agcctttctg ggcactgaat ccatcctgac ccaccacaca ccccccggcc
agttggcaag 2740 aaagaggtgg ctcttggggg cttttatgcc cttcattagc
tgatgttgga ttttatatgc 2800 atttttatat tgtctctaag tgtcagaact
ataatttatt catttctctg tgtgtgtgtg 2860 tgccaagaaa cgcaggctct
gggcctgcct ccttgcccag gaggccttgc cagcctgtgt 2920 gcttgtgaga
acacattgta cctgagctga caggtaccaa taaagacact ctatttttaa 2980
aaaaaaaaaa aaaaa 2995 27 581 PRT Eukaryote 27 Met Ala Gly Ile Gln
Lys Arg Lys Phe Asp Gln Leu Glu Glu Asp Asp 1 5 10 15 Cys Ser Ser
Ser Ser Leu Ser Ser Gly Asp Leu Ser Pro Ser Pro Pro 20 25 30 Ser
Ser Ser Ala Ser Pro Ala Trp Thr Ser Glu Glu Glu Gly Leu Gly 35 40
45 Asp Gln Pro Pro Gln Pro Asp Gln Asp Ser Ser Gly Ile Gln Ser Leu
50 55 60 Thr Pro Pro Ser Ile Leu Lys Arg Ala Pro Arg Glu Arg Pro
Gly His 65 70 75 80 Val Ala Phe Asp Gly Ile Thr Val Tyr Tyr Phe Pro
Arg Cys Gln Gly 85 90 95 Phe Thr Ser Val Pro Ser His Gly Gly Cys
Thr Leu Gly Met Ala Ser 100 105 110 Arg His Ser Thr Cys Arg Leu Phe
Ser Leu Ala Glu Phe Lys Gln Glu 115 120 125 Gln Phe Arg Ala Arg Arg
Glu Lys Leu Arg Arg Arg Leu Lys Glu Glu 130 135 140 Lys Leu Glu Met
Leu Lys Trp Lys Leu Ser Val Ser Gly Val Pro Glu 145 150 155 160 Ala
Gly Ala Asp Val Pro Leu Thr Val Asp Ala Ile Asp Asp Ala Ser 165 170
175 Val Glu Glu Asp Leu Ala Val Ala Val Ala Gly Gly Arg Leu Glu Glu
180 185 190 Ala Asn Phe Leu Gln Pro Tyr Pro Pro Arg Gln Arg Arg Ala
Leu Leu 195 200 205 Arg Ala Ser Gly Val Arg Arg Ile Asp Arg Glu Glu
Lys His Glu Leu 210 215 220 Gln Ala Leu Arg Gln Ser Arg Glu Asp Cys
Gly Cys His Cys Asp Gly 225 230 235 240 Val Cys Asp Pro Glu Thr Cys
Ser Cys Ile Leu Ala Gly Ile Lys Cys 245 250 255 Gln Met Asp His Thr
Ser Phe Pro Cys Gly Cys Cys Ser Glu Gly Cys 260 265 270 Glu Asn Pro
His Gly Arg Val Glu Phe Asn Gln Ala Arg Val Gln Thr 275 280 285 His
Phe Ile His Thr Leu Thr Arg Leu Gln Met Glu Gln Gly Ala Glu 290 295
300 Ser Leu Gly Asp Pro Glu Ser Pro Met Glu Asp Val Pro Val Glu Gln
305 310 315 320 Thr Val Val Ser Pro Phe Pro Pro Ser Lys Pro Thr Met
Ser Asn Asp 325 330 335 Leu Gly Asp Ser Ser Cys Gly Ser Asp Met Thr
Asp Ser Ser Thr Thr 340 345 350 Tyr Ser Ser Gly Gly Ser Gly Ser Arg
Ser Glu Ala Pro Asn His Leu 355 360 365 Ala His Pro Ser Leu Pro Gly
Ser Ser Phe Arg Ser Gly Ile Asp Glu 370 375 380 Asp Ser Leu Glu Gln
Ile Leu Asn Phe Ser Asp Ser Asp Leu Gly Ile 385 390 395 400 Glu Glu
Glu Glu Glu Glu Gly Gly Ser Val Gly Asn Leu Asp Asn Leu 405 410 415
Ser Cys Phe His Leu Ala Asp Ile Phe Gly Thr Gly Asp Pro Gly Ser 420
425 430 Leu Ala Ser Trp Thr His Ser Gln Phe Gly Ser Ser Leu Pro Ser
Gly 435 440 445 Ile Leu Asp Glu Asn Ala Asn Leu Asp Ala Ser Cys Phe
Leu Ser Ser 450 455 460 Gly Leu Glu Gly Leu Arg Glu Gly Ser Leu Pro
Ser Ser Ser Gly Ser 465 470 475 480 Pro Glu Gly Glu Ala Ala Gln Ser
Ser Ser Leu Asp Leu Ser Leu Ser 485 490 495 Ser Cys Asp Ser Phe Glu
Leu Leu Gln Ser Leu Pro Asp Tyr Ser Leu 500 505 510 Gly Pro His Tyr
Thr Ser Arg Arg Val Ser Gly Ser Leu Asp Ser Leu 515 520 525 Glu Thr
Phe His Pro Ser Pro Ser Phe Ser Pro Pro Arg Asp Ala Ser 530 535 540
Phe Leu Asp Ser Leu Ile Gly Leu Ser Glu Pro Val Thr Asp Val Leu 545
550 555 560 Ala Pro Leu Leu Glu Ser Gln Phe Glu Asp Thr Ala Val Val
Pro Leu 565 570 575 Asp Pro Val Pro Val 580 28 2938 DNA Eukaryote
28 attcggcacg agccagagtg aaggggcatg gagaagtgga cggcctggga
gccgcagggc 60 gccgatgcgc tgcggcgctt tcaagggttg ctgctggacc
gccgcggccg gctgcactgc 120 caagtgttgc gcctgcgcga agtggcccgg
aggctcgagc gtctacggag gcgctccttg 180 gcagccaacg tagctggcag
ctctctgagc gctgctggcg ccctagcagc catcgtgggg 240 ttatcactca
gcccggtcac cctgggagcc tcgctcgtgg cgtccgccgt gggcttaggg 300
gtggccaccg ccggaggggc agtcaccatc acgtccgacc tctctctgat cttctgcaat
360 tcccgggagg tacggagggt gcaagagatc gccgccacct gccaggacca
gatgcgcgaa 420 ctcctgagct gccttgagtt cttctgtcag tggcaggggc
gcggggaccg ccagctgctg 480 cagagcggga gggacgcctc catggctctt
tacaactctg tctacttcat cgtcttcttc 540 ggctcgcgtg gcttcctcat
ccccaggcgt gcggaggggg ccaccaaagt cagccaggcc 600 gtgctgaagg
ccaagattca gaaactgtct gagagcctgg agtcctgcac tggtgccctg 660
gatgaactta gtgagcagct ggaatcccgg gtccagctct gtaccaaggc cggccgtggt
720 cacaacctca ggaactcccc tgatctggat gcagcgttgt ttttctaaga
gcatcctcta 780 gctgtgtgga atgttctaga ttcgcagcat ccacaaggaa
gtgctacatg ggcggagtgc 840 aaaggatttc agaagctctt cttgcagggc
atcagtccgt agctccttgt gtgtgcgaaa 900 gacttttcac ttgtgtaatc
ccaactgagt atgtgaccct aaacagtcac tttggggact 960 ccccaaatcc
tttttagctg cacacagctt gtcagactgt ccttcaatta gagttattgg 1020
ggtggggggg cttgatggct tgagtaatag aggtctggcg aggtgtctcc ctcttggacc
1080 tcttatgtgt tgttactaga atcctgagat tctcaaatgt tggtgagagg
agacttttac 1140 ttttcaactt tgcttcggca gtttccgata cacaggactc
cagaatccag aacaagaaag 1200 aagaaccttg tgtttgtagg gtgtgcagac
ccagacgggg ccgaggagct gacttgctca 1260 gctctcacac gcagccagtt
tatccactca cagaccaaac ctggctactg catagactgt 1320 tccagtgtgg
cttcaaatcc acacctctag gtaccctgag aaggaaagcc acctgaagag 1380
tcactctaat cccaacacgc tcaccccctt cacgtccata aaggagctgg gcaaggggtg
1440 agatgaagac cctgacaatt ttaaatgact gtagcataga gagccatggc
ctttgagttt 1500 aagagtcttg atcccaggtt ctgtccccca ctgtcctgtg
acttagccac cttgtcttgc 1560 tacagatggt ggtaggaggc caccctgttg
cgaagtcctg agataatgac aaacacagag 1620 gctagctcac aaaaatgtac
ttcctggcct ggcttctgaa gggttaactg ttgggctcca 1680 tcccagattt
ctgagatcag gaactccaaa tatgaggccc gcctctggct gattctgatg 1740
ccccataaat gtttgaaaat gacacagcaa aggttcatct ccagccaggt gtggtgggac
1800 acacctgtaa ggccagcgct tggagatgga gacaggggga ccagtagttc
agggtcattc 1860 ttggctacat agcaaactca aggccaccct ggtctcaaaa
accaaaacaa aaagccatct 1920 tctgactccc ttcaattgtt caaagccttt
ccagggcctt cagaatcacg ctcagagtgt 1980 tctgggaaga ttagcccaga
agccagagaa agagtacgct gtgtgcttgt aaagccagtt 2040 actctgtccc
ctgtgaacta ggagacagag cacttccgac cctatagagg gcagtagtgg 2100
ccattccttg taggggactg gtatagaagt aatgtgaact atttaaaaat agttatttaa
2160 ttgctgcctt cacatttgat
tttatttaac cttcacatta tttagaaaat aataagagta 2220 gtaagtgtct
gaataggaag ggagtctctt aaggctcttt ccaagagctc aggtttggat 2280
ttctagagtc cccccgaccc cagagaggac tctttagtgt ttgacacggt ctttgtaagt
2340 aagatgggga gtcctggaga gagagaccaa gctgattttt aaactaggaa
atggagtctt 2400 gaactgtgga agatttgaaa agttaagcct atgtgtcttg
aaggtacttg gccagaaaag 2460 cacttggctt gaaaaagaaa acctgtttaa
ttcaggggtg gaggaataga gacagatgaa 2520 gaaagcattt agacctcgga
aacctgatgt cctatgaaat tctgttttta taaaattgtg 2580 ttatggtgga
gatctgttgc atttcgactt tgtggctgta agaaacctgt tatctatgtt 2640
taagaaagta cttctaattt attcaatgtc ttcctaaatt atcctttaaa aaaaaaagtt
2700 ggaaagtcta tgagaccgta cctaagaaac cttgactgtg tatttaagtt
atttaatgcc 2760 atgcatttgt gaagcccctt cccagtgatg gctgtggtgt
gtctgaggaa atgtaagttt 2820 ggcatgaggg ggaggggctg ctgtttctat
atttgttttt gttttctata aacagtaatc 2880 aggatgtatc ctggtttcat
ttgacattga aaaaaaaaaa aaaactcgtg ccgaattc 2938 29 2527 DNA
Eukaryote CDS (41)...(871) 29 tacggctgcg agaagacgac agaaggggag
cggagccaag atg gcg gcg gag ctg 55 Met Ala Ala Glu Leu 1 5 gaa tac
gag tct gtg ctg tgt gtg aag ccc gac gtc agc gtc tac cgg 103 Glu Tyr
Glu Ser Val Leu Cys Val Lys Pro Asp Val Ser Val Tyr Arg 10 15 20
att ccg ccg cgg gcc tcc aac cgc ggt tac agg gca tct gac tgg aag 151
Ile Pro Pro Arg Ala Ser Asn Arg Gly Tyr Arg Ala Ser Asp Trp Lys 25
30 35 cta gac cag cct gat tgg act ggt cgc ctc cga atc act tca aaa
ggg 199 Leu Asp Gln Pro Asp Trp Thr Gly Arg Leu Arg Ile Thr Ser Lys
Gly 40 45 50 aag att gcc tac atc aaa ctg gaa gat aaa gtt tca ggg
gag ctc ttc 247 Lys Ile Ala Tyr Ile Lys Leu Glu Asp Lys Val Ser Gly
Glu Leu Phe 55 60 65 gct cag gcg cca gta gag cag tac cct ggg att
gct gtg gag act gtg 295 Ala Gln Ala Pro Val Glu Gln Tyr Pro Gly Ile
Ala Val Glu Thr Val 70 75 80 85 gcc gac tcc agc cgc tac ttt gtg atc
agg atc cag gat ggc acc ggg 343 Ala Asp Ser Ser Arg Tyr Phe Val Ile
Arg Ile Gln Asp Gly Thr Gly 90 95 100 cgc agt gcg ttt att ggc atc
ggc ttc acg gac cgg gga gat gcc ttc 391 Arg Ser Ala Phe Ile Gly Ile
Gly Phe Thr Asp Arg Gly Asp Ala Phe 105 110 115 gac ttt aat gtc tcc
ctg caa gat cac ttc aag tgg gta aag cag gaa 439 Asp Phe Asn Val Ser
Leu Gln Asp His Phe Lys Trp Val Lys Gln Glu 120 125 130 acc gag atc
tcc aaa gaa tcg cag gaa atg gat agt cgt ccc aag ttg 487 Thr Glu Ile
Ser Lys Glu Ser Gln Glu Met Asp Ser Arg Pro Lys Leu 135 140 145 gat
tta ggc ttc aag gaa ggg caa acc atc aag ctg agt att ggg aac 535 Asp
Leu Gly Phe Lys Glu Gly Gln Thr Ile Lys Leu Ser Ile Gly Asn 150 155
160 165 att aca gcc aag aaa ggg ggt act tct aag ccc cgg gcc tca gga
acg 583 Ile Thr Ala Lys Lys Gly Gly Thr Ser Lys Pro Arg Ala Ser Gly
Thr 170 175 180 ggg ggc ctg agc tta ctc cca cct cct cct gga ggc aaa
gtc act atc 631 Gly Gly Leu Ser Leu Leu Pro Pro Pro Pro Gly Gly Lys
Val Thr Ile 185 190 195 ccc cca ccg tcc tcc tcc gtt gcc atc agc aac
cac gtc acc cca cca 679 Pro Pro Pro Ser Ser Ser Val Ala Ile Ser Asn
His Val Thr Pro Pro 200 205 210 ccc att cca aaa tct aac cat gga agt
aat gat tca gat atc ctg tta 727 Pro Ile Pro Lys Ser Asn His Gly Ser
Asn Asp Ser Asp Ile Leu Leu 215 220 225 gat ttg gat tct cca gct cct
gtc ccg act tca gca cca gct cca gct 775 Asp Leu Asp Ser Pro Ala Pro
Val Pro Thr Ser Ala Pro Ala Pro Ala 230 235 240 245 cca gct tct aca
agc aat gac ttg tgg gga gac ttt agc act gca tcc 823 Pro Ala Ser Thr
Ser Asn Asp Leu Trp Gly Asp Phe Ser Thr Ala Ser 250 255 260 agc tct
gtt cca aac cag gca cca cag cca tct aac tgg gtc cag ttt 871 Ser Ser
Val Pro Asn Gln Ala Pro Gln Pro Ser Asn Trp Val Gln Phe 265 270 275
tgagtcgcat tggcaagaag ttgaggacac ttgaagaata aaaatgacct caagggcacc
931 attctatgag ggagttgagg gacggcttaa tttcccagga cccaaatcag
tggtcagtct 991 ttcctgtagc ttctctgtgc attcaggctg gatttttttt
tttttttttt ttggttacct 1051 ctgtgttact tgctgtatat ccaggagaca
atctgctgtt tcctgctcag aaccaagcaa 1111 gggagtagtg ggtattatca
cactgactga ctttgcagag ttcagaaggc caacttgatg 1171 agtgggagtg
acctcgaacg tatgtaaatc cttgaactta tttcagaatc atctcatgat 1231
tccctagtta gcaatttcag gagagacaaa tgccttgaaa ctgtcttctc cactaatccg
1291 agactaaata tggtcaggct ggccccagga ctcatgaagt tagggttttc
atgggggtag 1351 atttggagaa agctgtgtcc cggctctctt ctgtaaggcc
tccttcaggc ttaccccatg 1411 cagtgaactt cccgtgctgg gtggagcccc
atcaccttct tgtgtgttta catgttgttt 1471 cctttgacaa gagggttatg
ttggtggcac ctcactgttt tcttgttgaa tagtgcagca 1531 tctttgacca
gtgaatattt ctgagatgaa ggggtcaagg ggctgtgctt tccatggtgt 1591
agtctacaga agtgtttaat ttcttgcggc cccacgggat tgctgcactg acgcatagaa
1651 ttgatctata ctcaccctgt gtttgacctg aagagtttta acttgatgtg
tagagcagag 1711 agctggaagc actaagttcc cattcagtac ccacaatgcc
ttgctgcctg gtttgactcc 1771 ttttcataaa catttcattt cagtccatct
agcacttctg tggaaagctg ctgttgattg 1831 tgtcagtgtg aaggaggtga
agtcacagct ttctttacct atgacagtta ggctttgcac 1891 tagacgttga
taccagctag gatatcttaa aggaagttac cgccccatca ctctccagtc 1951
tctggccgcc attcctttta cagtgctgtg aagagcgtcc tctgaggtcg gtgggtactg
2011 tctcctgttg gtcgggcagt ttgagggagg agtgggagga ctcacactcc
tgcaggtacc 2071 tgtttgggta gcacactggc tgcagagagt cctttcagat
atattgtttc tcaatgttct 2131 tcgtagcttt ttctaacttc gggtccattt
ttcccatcgc ctcttcccat tcccaggcag 2191 ctctcttgtt gcagagccat
ggcaggacgt ttaagttcca ataaaaacac taagaagaaa 2251 gtatagaatc
actagtgact gttgggaaac ctattttctc aatcttcctc cattttgtgt 2311
tctttgtatt cttaagatga taatatatta tgtatttgaa ttgctgaaaa ttgaaaatga
2371 agttgaagat atatgtatat aagcgtatgc tgtattggtg caataatggt
aattaaagat 2431 attaaaaaag aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa 2491 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaa
2527 30 277 PRT Eukaryote 30 Met Ala Ala Glu Leu Glu Tyr Glu Ser
Val Leu Cys Val Lys Pro Asp 1 5 10 15 Val Ser Val Tyr Arg Ile Pro
Pro Arg Ala Ser Asn Arg Gly Tyr Arg 20 25 30 Ala Ser Asp Trp Lys
Leu Asp Gln Pro Asp Trp Thr Gly Arg Leu Arg 35 40 45 Ile Thr Ser
Lys Gly Lys Ile Ala Tyr Ile Lys Leu Glu Asp Lys Val 50 55 60 Ser
Gly Glu Leu Phe Ala Gln Ala Pro Val Glu Gln Tyr Pro Gly Ile 65 70
75 80 Ala Val Glu Thr Val Ala Asp Ser Ser Arg Tyr Phe Val Ile Arg
Ile 85 90 95 Gln Asp Gly Thr Gly Arg Ser Ala Phe Ile Gly Ile Gly
Phe Thr Asp 100 105 110 Arg Gly Asp Ala Phe Asp Phe Asn Val Ser Leu
Gln Asp His Phe Lys 115 120 125 Trp Val Lys Gln Glu Thr Glu Ile Ser
Lys Glu Ser Gln Glu Met Asp 130 135 140 Ser Arg Pro Lys Leu Asp Leu
Gly Phe Lys Glu Gly Gln Thr Ile Lys 145 150 155 160 Leu Ser Ile Gly
Asn Ile Thr Ala Lys Lys Gly Gly Thr Ser Lys Pro 165 170 175 Arg Ala
Ser Gly Thr Gly Gly Leu Ser Leu Leu Pro Pro Pro Pro Gly 180 185 190
Gly Lys Val Thr Ile Pro Pro Pro Ser Ser Ser Val Ala Ile Ser Asn 195
200 205 His Val Thr Pro Pro Pro Ile Pro Lys Ser Asn His Gly Ser Asn
Asp 210 215 220 Ser Asp Ile Leu Leu Asp Leu Asp Ser Pro Ala Pro Val
Pro Thr Ser 225 230 235 240 Ala Pro Ala Pro Ala Pro Ala Ser Thr Ser
Asn Asp Leu Trp Gly Asp 245 250 255 Phe Ser Thr Ala Ser Ser Ser Val
Pro Asn Gln Ala Pro Gln Pro Ser 260 265 270 Asn Trp Val Gln Phe 275
31 2922 DNA Eukaryote CDS (28)...(768) 31 attcggcacg agccagagtg
aaggggc atg gag aag tgg acg gcc tgg gag ccg 54 Met Glu Lys Trp Thr
Ala Trp Glu Pro 1 5 cag ggc gcc gat gcg ctg cgg cgc ttt caa ggg ttg
ctg ctg gac cgc 102 Gln Gly Ala Asp Ala Leu Arg Arg Phe Gln Gly Leu
Leu Leu Asp Arg 10 15 20 25 cgc ggc cgg ctg cac tgc caa gtg ttg cgc
ctg cgc gaa gtg gcc cgg 150 Arg Gly Arg Leu His Cys Gln Val Leu Arg
Leu Arg Glu Val Ala Arg 30 35 40 agg ctc gag cgt cta cgg agg cgc
tcc ttg gca gcc aac gta gct ggc 198 Arg Leu Glu Arg Leu Arg Arg Arg
Ser Leu Ala Ala Asn Val Ala Gly 45 50 55 agc tct ctg agc gct gct
ggc gcc cta gca gcc atc gtg ggg tta tca 246 Ser Ser Leu Ser Ala Ala
Gly Ala Leu Ala Ala Ile Val Gly Leu Ser 60 65 70 ctc agc ccg gtc
acc ctg gga gcc tcg ctc gtg gcg tcc gcc gtg ggc 294 Leu Ser Pro Val
Thr Leu Gly Ala Ser Leu Val Ala Ser Ala Val Gly 75 80 85 tta ggg
gtg gcc acc gcc gga ggg gca gtc acc atc acg tcc gac ctc 342 Leu Gly
Val Ala Thr Ala Gly Gly Ala Val Thr Ile Thr Ser Asp Leu 90 95 100
105 tct ctg atc ttc tgc aat tcc cgg gag gta cgg agg gtg caa gag atc
390 Ser Leu Ile Phe Cys Asn Ser Arg Glu Val Arg Arg Val Gln Glu Ile
110 115 120 gcc gcc acc tgc cag gac cag atg cgc gaa ctc ctg agc tgc
ctt gag 438 Ala Ala Thr Cys Gln Asp Gln Met Arg Glu Leu Leu Ser Cys
Leu Glu 125 130 135 ttc ttc tgt cag tgg cag ggg cgc ggg gac cgc cag
ctg ctg cag agc 486 Phe Phe Cys Gln Trp Gln Gly Arg Gly Asp Arg Gln
Leu Leu Gln Ser 140 145 150 ggg agg gac gcc tcc atg gct ctt tac aac
tct gtc tac ttc atc gtc 534 Gly Arg Asp Ala Ser Met Ala Leu Tyr Asn
Ser Val Tyr Phe Ile Val 155 160 165 ttc ttc ggc tcg cgt ggc ttc ctc
atc ccc agg cgt gcg gag ggg gcc 582 Phe Phe Gly Ser Arg Gly Phe Leu
Ile Pro Arg Arg Ala Glu Gly Ala 170 175 180 185 acc aaa gtc agc cag
gcc gtg ctg aag gcc aag att cag aaa ctg tct 630 Thr Lys Val Ser Gln
Ala Val Leu Lys Ala Lys Ile Gln Lys Leu Ser 190 195 200 gag agc ctg
gag tcc tgc act ggt gcc ctg gat gaa ctt agt gag cag 678 Glu Ser Leu
Glu Ser Cys Thr Gly Ala Leu Asp Glu Leu Ser Glu Gln 205 210 215 ctg
gaa tcc cgg gtc cag ctc tgt acc aag gcc ggc cgt ggt cac aac 726 Leu
Glu Ser Arg Val Gln Leu Cys Thr Lys Ala Gly Arg Gly His Asn 220 225
230 ctc agg aac tcc cct gat ctg gat gca gcg ttg ttt ttc taa 768 Leu
Arg Asn Ser Pro Asp Leu Asp Ala Ala Leu Phe Phe * 235 240 245
gagcatcctc tagctgtgtg gaatgttcta gattcgcagc atccacaagg aagtgctaca
828 tgggcggagt gcaaaggatt tcagaagctc ttcttgcagg gcatcagtcc
gtagctcctt 888 gtgtgtgcga aagacttttc acttgtgtaa tcccaactga
gtatgtgacc ctaaacagtc 948 actttgggga ctccccaaat cctttttagc
tgcacacagc ttgtcagact gtccttcaat 1008 tagagttatt ggggtggggg
ggcttgatgg cttgagtaat agaggtctgg cgaggtgtct 1068 ccctcttgga
cctcttatgt gttgttacta gaatcctgag attctcaaat gttggtgaga 1128
ggagactttt acttttcaac tttgcttcgg cagtttccga tacacaggac tccagaatcc
1188 agaacaagaa agaagaacct tgtgtttgta gggtgtgcag acccagacgg
ggccgaggag 1248 ctgacttgct cagctctcac acgcagccag tttatccact
cacagaccaa acctggctac 1308 tgcatagact gttccagtgt ggcttcaaat
ccacacctct aggtaccctg agaaggaaag 1368 ccacctgaag agtcactcta
atcccaacac gctcaccccc ttcacgtcca taaaggagct 1428 gggcaagggg
tgagatgaag accctgacaa ttttaaatga ctgtagcata gagagccatg 1488
gcctttgagt ttaagagtct tgatcccagg ttctgtcccc cactgtcctg tgacttagcc
1548 accttgtctt gctacagatg gtggtaggag gccaccctgt tgcgaagtcc
tgagataatg 1608 acaaacacag aggctagctc acaaaaatgt acttcctggc
ctggcttctg aagggttaac 1668 tgttgggctc catcccagat ttctgagatc
aggaactcca aatatgaggc ccgcctctgg 1728 ctgattctga tgccccataa
atgtttgaaa atgacacagc aaaggttcat ctccagccag 1788 gtgtggtggg
acacacctgt aaggccagcg cttggagatg gagacagggg gaccagtagt 1848
tcagggtcat tcttggctac atagcaaact caaggccacc ctggtctcaa aaaccaaaac
1908 aaaaagccat cttctgactc ccttcaattg ttcaaagcct ttccagggcc
ttcagaatca 1968 cgctcagagt gttctgggaa gattagccca gaagccagag
aaagagtacg ctgtgtgctt 2028 gtaaagccag ttactctgtc ccctgtgaac
taggagacag agcacttccg accctataga 2088 gggcagtagt ggccattcct
tgtaggggac tggtatagaa gtaatgtgaa ctatttaaaa 2148 atagttattt
aattgctgcc ttcacatttg attttattta accttcacat tatttagaaa 2208
ataataagag tagtaagtgt ctgaatagga agggagtctc ttaaggctct ttccaagagc
2268 tcaggtttgg atttctagag tccccccgac cccagagagg actctttagt
gtttgacacg 2328 gtctttgtaa gtaagatggg gagtcctgga gagagagacc
aagctgattt ttaaactagg 2388 aaatggagtc ttgaactgtg gaagatttga
aaagttaagc ctatgtgtct tgaaggtact 2448 tggccagaaa agcacttggc
ttgaaaaaga aaacctgttt aattcagggg tggaggaata 2508 gagacagatg
aagaaagcat ttagacctcg gaaacctgat gtcctatgaa attctgtttt 2568
tataaaattg tgttatggtg gagatctgtt gcatttcgac tttgtggctg taagaaacct
2628 gttatctatg tttaagaaag tacttctaat ttattcaatg tcttcctaaa
ttatccttta 2688 aaaaaaaaag ttggaaagtc tatgagaccg tacctaagaa
accttgactg tgtatttaag 2748 ttatttaatg ccatgcattt gtgaagcccc
ttcccagtga tggctgtggt gtgtctgagg 2808 aaatgtaagt ttggcatgag
ggggaggggc tgctgtttct atatttgttt ttgttttcta 2868 taaacagtaa
tcaggatgta tcctggtttc atttgacatt gaaaaaaaaa aaaa 2922 32 246 PRT
Eukaryote 32 Met Glu Lys Trp Thr Ala Trp Glu Pro Gln Gly Ala Asp
Ala Leu Arg 1 5 10 15 Arg Phe Gln Gly Leu Leu Leu Asp Arg Arg Gly
Arg Leu His Cys Gln 20 25 30 Val Leu Arg Leu Arg Glu Val Ala Arg
Arg Leu Glu Arg Leu Arg Arg 35 40 45 Arg Ser Leu Ala Ala Asn Val
Ala Gly Ser Ser Leu Ser Ala Ala Gly 50 55 60 Ala Leu Ala Ala Ile
Val Gly Leu Ser Leu Ser Pro Val Thr Leu Gly 65 70 75 80 Ala Ser Leu
Val Ala Ser Ala Val Gly Leu Gly Val Ala Thr Ala Gly 85 90 95 Gly
Ala Val Thr Ile Thr Ser Asp Leu Ser Leu Ile Phe Cys Asn Ser 100 105
110 Arg Glu Val Arg Arg Val Gln Glu Ile Ala Ala Thr Cys Gln Asp Gln
115 120 125 Met Arg Glu Leu Leu Ser Cys Leu Glu Phe Phe Cys Gln Trp
Gln Gly 130 135 140 Arg Gly Asp Arg Gln Leu Leu Gln Ser Gly Arg Asp
Ala Ser Met Ala 145 150 155 160 Leu Tyr Asn Ser Val Tyr Phe Ile Val
Phe Phe Gly Ser Arg Gly Phe 165 170 175 Leu Ile Pro Arg Arg Ala Glu
Gly Ala Thr Lys Val Ser Gln Ala Val 180 185 190 Leu Lys Ala Lys Ile
Gln Lys Leu Ser Glu Ser Leu Glu Ser Cys Thr 195 200 205 Gly Ala Leu
Asp Glu Leu Ser Glu Gln Leu Glu Ser Arg Val Gln Leu 210 215 220 Cys
Thr Lys Ala Gly Arg Gly His Asn Leu Arg Asn Ser Pro Asp Leu 225 230
235 240 Asp Ala Ala Leu Phe Phe 245 33 1446 DNA Eukaryote
misc_feature (1)...(1446) n = A,T,C or G 33 gctttggaaa ccggactgca
ggctaaactg gcttcttttg aatccttgga agcataaagg 60 acaagtagca
gggctcgcag tcttccattt gtcactggag aagaacttat aattcagaag 120
atctgggtct ggacccaggc tgaccacttt ggagctttga gactctggga ttgtgatcca
180 gttctgagct ggtgataaac actccttgtg acttttggtc aattcagcta
ccagattcca 240 gccaacatga ccctcgcagc ctataaggag aagatgaagg
aactcccact agtgtctctg 300 ttctgctcct gttttctgtc tgatcccctg
aataaatcat cctacaaata tgaaggctgg 360 tgtgggagac agtgtaggag
gaaaggtcaa agccagcgga aaggcagtgc tgactggaga 420 gaaagaagag
aacaggcaga tacggtagac ctgaactggt gtgtcatctc tgatatggaa 480
gtcatcgagc tgaataagtg tacctcgggc cagtcctttg aagtcatcct gaagccacct
540 tcctttgacg gggtgcctga gtttaatgcc tccctcccaa gacgtcgaga
cccatcgcta 600 gaagagatac agaagaagct agaagcagca gaggagcgaa
ggaagtacca ggaagctgag 660 ctcctaaaac accttgcaga gaaacgagag
catgagcgtg aggtaatcca gaaagctatc 720 gaggaaaaca acaacttcat
caagatggcg aaagagaagc tggcccagaa gatggagtcc 780 aataaggaaa
accgggaggc ccatctggct gccatgttgg agcggctgca agagaaggac 840
aagcacgcag aggaggtgcg gaaaaacaag gagctgaagg aagaggcctc caggtaaagc
900 ccanaggcca aggaagtttc caggacagcc ggacagctcc cgcagcaacc
tggttccagc 960 agcatcggcc gctggctgct ctcccagcac tggggttcgg
ggggaggggg gtggccaaag 1020 gggcgtttcc tctgcttttg gtgtttgtac
atgtaaaaga ttgaccagtg aagccatcct 1080 atttgtttct ggggaacaat
gatggggtgg gagaggggac agagagtgtt tggaaaagga 1140 ggtgaagatg
agcccgagga ctttgtgaca ctgtccactg actgcagact tgggccaagg 1200
cccccgcttt tcacggctct gcctggacat tcggcctcca ggttcctagt ggagagaaga
1260 tgtgacagaa gttcagagtg aagggccgag tcctggtggg gtggtgtgca
gggccagcag 1320 gacgagcccg tctggatgga gtgaaaccta ccctgagcgg
gtgggataag gtctgtgtgc 1380 gtctgttcat tgtcatcttt tgatcatcat
gaccaacgaa acatttaaaa aaaaaaaaaa 1440 aaaaaa 1446 34 5305 DNA
Eukaryote misc_feature (1)...(5305) r = G or A y = C or T m = A or
C k = G or T s = G or C w = A or T n = A, T, G, or C 34 gataaacact
ccttgtgact tttggtcaat tcagctacca gattccagcc aacatgaccc 60
tcgcaggtag gtacatgcac cagtcagtga tgaacaccat aacacaagcc atttttctat
120 ctctgtgtgt gtccatgtgt attaaggtgc atccgtgtgt gtgatacaca
cgtaggtgca 180 tggcatgcat gtgtgtgcaa atgcatatac aagtccaagg
acaggggttg gggatttagc 240 tcantggtag agcacttgcc tangaagcgc
aaggccctgg gttcggtccc cagctccgaa 300 aaaaagaacc aaaaaaaaaa
aaaaaaaaaa aaaaaaaatt tccanggaca accccaaatt 360 tcctttcncn
aaanccancc ancttccatt naaaaaaaan gggtcncncn tgggttaaac 420
catttnnaaa nggcnaacct nacnggccak tgaktgccag gaatcttctt atycctgccc
480 wacctccaat gtctttcaca tgtgaatgct gagggtcaga acttgtgctt
acaaggcaga 540 cattttgcca gctctccggc catctttctc tatgtatgta
cactcacaga tgcacaggaa 600 gagagggtag agaagccaag aggcaaagtc
atttctgggt ggtgggtggg atcacagctg 660 aattcttctt cctcatttgc
tctgtgtgta ttatttaatt ttaaaataat acctttataa 720 tagtatcgaa
actatgcttt caagtttgta agagaaagtg atcactgggc tgtgtagtga 780
gggggtcttt atattatgca tataacatgg tgcaatggga aggactggca gaggcctcca
840 tgatgaccta tgacttctag ggagactcag tcgtgtcaag ggtacattcc
tactctgcag 900 acagcttctc cctggtttga ttcctgtgct gggaagattt
gaggagtctt ccagcctgac 960 ctcttctaca gtgggcctgg actttaagga
gagtagcaag gaagtctttt tattaatctc 1020 ttacccttta ggcagcagtg
tcaagtactt ttagcagaat taaatataga tttcctacaa 1080 actacaaact
tcaaagccct ggtttatcct tgggtgggag taggagatgg agggccaggg 1140
tcagggcact gcacttggga tctttacttg agggtactca acgcttggta gtaacaaaaa
1200 gtggggtgag tgacaatgtt aattttcaac tgggaggtag cccaggcttg
ggtactttgg 1260 agccagaaag cctgggctga ctcacagaag tggtgctctc
tctygyagcc tataaggagw 1320 wgatgaagga actcccacta gtgtctctgt
tctgctcctg ttttctgtct gatccccygr 1380 ataaatcatc ctacaaatat
gaaggtgagt aggggctagg ctgggataga aaagggtgga 1440 ggcttctgtg
tcctgtgttt gtsggtgccc cacattgact cctatcttgt aaaactgtcc 1500
tggtcgcagt gtgtcttatt tcccagaggc tgaggagtct gagcccaggg ggatgtagcc
1560 tgggtgccaa gcagcctcca gggatctgga ttgggccctc ctggagcact
tgctcctaga 1620 gtcccttttr cacattcctt gacaccacag aggacaccag
gataagccag acacaagttt 1680 tgagattcca ttcatggagg cccagaacag
aaaaagaaaa cttagtgtgt tcaccagggc 1740 ttctagggac aggtagagat
gctcctagac aggtccaggg tgggaatagc acttctaacc 1800 tggatggtga
cagttcgagc ccctagaccc tatcagagag tactggattg tcatgctgtc 1860
aggaggagtg gtcaggggac agataggtca tctcttcatt tctgtttgcc aggaagggat
1920 gggtttggtc tgtcaataag agagatgggt gtttggatga cctgagtctg
ttttttccat 1980 ttaggctggt gtgggagaca gtgtaggagg aaaggtcaaa
gccagcggaa aggcagtgct 2040 gactggagag aaagaagaga acagggtagg
ccggagccag gggagaggtc cacaagccat 2100 cagagggaca gggcaaggag
gggctggcgg tggggatggg tgaaatgaac tggtgtctgt 2160 caccagcgag
gaacaacagc agctggtgct atcacaaatc acagctccct gcttaccctg 2220
taaaagccat tgaccttagg gtccaacgtt caggatcgac cagaccccta gtcattggtg
2280 tgccttggga ccctcagctt tcctgtgtct gtgtgcatgt acacatgctc
attggggccc 2340 cagctgctcc tcagaaggtg agcagcccca actctgccct
ccatagcaga tacggtagac 2400 ctgaactggt gtgtcatctc tgatatggaa
gtcatcgagc tgagtaagtg tacctcgggc 2460 cagtcctttg aagtcatcct
gaagccacct tcctttgacg gggtgcctga gtttaaagcc 2520 tccctcccaa
gacgtcgaga cccatcgcta gaagagatac agaagaagct agaagcagca 2580
gaggagcgaa ggaaggttag tgtagcccca tgtcacttcc tcccatccca gcgggagcag
2640 gaagtgcagc tccatatctc ttcctcccat cccagtggga gtgggaagga
tatttagaca 2700 gcacctcctg agtgctgggc atagaccggt agttctcaac
cttcttagtg ctgtaaccct 2760 taatatatat atatatatat atatatatat
atatatatat agttcctcat gttgtgatta 2820 ccccccatac cataaactta
tcccgttgct ctttatgtct tcataattat aattttgcta 2880 ctgttatgaa
ttgtgataca actatcagac ctgcaccccc taatggcagc agcccacgtg 2940
ttgagaacca ctggcataga tgtagactaa gataccacct gaaggggaca agactatgac
3000 tatgcactgg gtgagcttac agtgtggcta atggctaaaa tgtcacagtc
ctcacaaagc 3060 tgcctttgta tgcagcttcc ttgttcccca ttgattctmg
tccstcagct cagatgccca 3120 ttttaatgtg agtgtttctt nacctttcag
aaanacaaaa caaaacaacc cagctttctc 3180 cactnaattg tgtggtccct
ccctttaaat atccaaagca tttatcacac ccaggtctgg 3240 ngtccantat
ntattgatat gcgtgtttat ttnnactagg gcaattntct ccnttccctg 3300
gtgtctggag ttgtgagggc cttgaggttt atagaagatc acttagtact tgtgaatgaa
3360 cgcgaggaaa aggagaaaag agactcagaa gctacttngg aaagggctac
naaagccaaa 3420 tatgacggaa aggtttgcag tccatgncgt tgttctctgc
ttctgggaca gaggaccagg 3480 ttcatctcat ctgggcatgg cactgttcag
ctgtggtggt agaaatccac tctaaagggt 3540 cnttctcttt cttttgntgc
cctagtacca ggaagctgag ctcctaaaac accttgcaga 3600 gaaacgagag
catgagcgtg aggtaatcca gaaagctatc gaggaaaaca acaacttcat 3660
caagatggcg aaagagaagc tggcccagaa gatggagtcc aataaggaaa accgggaggc
3720 ccatctggct gccatgttgg agcggctgca agagaaggta agaggtcctg
gattggcagg 3780 aggctccttc catggcaaga acgtgcaacc tacacatcac
tctggaggaa gcggcctatg 3840 caggaattga aatgtttcta ccaggcaggg
tcctcattgt tctaagggga agatttggga 3900 agtcataggc aagaagctca
caccaaaccc tgggtggcct ccggggatct tctanggttt 3960 tgaaccggaa
attctgcact gtctcangag cttgctcaca cccttctttt ctaaagaaag 4020
cccgcccagt gcaagtatct aaggagaggc acatgtctac acatttctgg cttcatcatt
4080 gaatgggcag atttgggtta gtgaaagata cagtcagctt ggctttgagc
canggataca 4140 gcaagctcgg ttgccaatac agcaggatac aggattctcc
ccagagctcc tcgtaagggc 4200 cagagagtan taggttttcc tcaatagtct
gcctttgtca ataactcaaa tgtcacctgc 4260 atctgagcgg tgtgcgagac
tggggttggt cctccatgtt attctttgga agacgtgctg 4320 acctcatttc
ctgagtccca ggctgcctac gtttctcctg cagctcctgg gaagctttag 4380
ctctgtgttt tatttccaag gagccgcctg ctgcgcggtg actcccggga csgatcggtg
4440 gcctcgtccc atggtgagca gcgtggtcct tattccttcc tgcctaccca
cctaaaacct 4500 caggcccttg acaattacca cagaaagatc tggcttcatc
cagggatgtg agcagcacag 4560 gctggccagt aggtggcagc cctgtgctca
tgttcaatta caggagggac agcaaggctt 4620 ctttctccac tgagtgcctt
gggggaggga cacaatctga gtgtgacttt gggctcctcc 4680 agttaatgag
agatactgta agaaaactta agattgcctt tactttttat accaggtctc 4740
atgcattcca ggctggcctc aaattggcta aattgctgag gctagccttg gaattcttat
4800 cattttgtct tcacctccaa gtgcagggat tacaggcatg tgctgccaag
cctattcaat 4860 gcaggtttgg ggcttgaacc cagggctctg tgcatgcaag
ctaggcactc tgccaacagt 4920 gccatagccc caactcaagg caaattcttg
aggaaaccac agatagaatg ggagagttat 4980 gggattgcag actcagctta
aaatacatca caaagttagg ttgtgttgaa gcacttgaat 5040 gtttgtttat
ataacgattc tattttatca taactcggtc atcacaagtt tacaaggcaa 5100
acattcttag tccagataag gaaaccattc tagaggtcaa atgattccag agattnacag
5160 ggtatacgac aatanattgg ccctggccnc taatcaatgg ctgcttcttg
ccgggtaaag 5220 aaaacatcca atataancca cnnctttcan agcaanaatt
tcaaagacaa caagcagggc 5280 aaaaccaggg tccaaagcaa ccact 5305 35 796
DNA Eukaryote misc_feature (1)...(796) n = A, T, G, or C 35
tgggcgggaa agcagtttgt cttgttgntg aattatgtta nnaagcaaat gaagttatct
60 tccaacacat gtgagggagt ccattgtctg gagtcaagca ntatttccca
acagttctct 120 gtcagtacat aacgcaaggt cctccttcag tcagagattt
aagacaacac taaagagatg 180 gagagaaata acacatctgt ggtgtgtcag
ggacgctggc aatgggctga tcttttccca 240 ttcnttntaa actggctgtc
ccaaagggcc cnttgtattt agtcaagtga ccattccaag 300 cgccagaatg
accagtggag gtgcagagag cntagggtgt cttggggtcg ctgtgaggtg 360
ggtcccctgc aggatgtcta tgcacttgca ggcttataca cctgtgtccc gcgtnttact
420 tgcctccttc cacccctctt aggatacctt cgccgacagc tctgctctgc
ccgtggtgac 480 catcttttgc gctccattct cttgcccttt gtcttcccct
ggcagccttg tgtgacccgc 540 ctttgtccct cccttcctct ccaggacaag
cacgcagagg aggtgcggaa aaacaaggag 600 ctgaaggaag aggcctccag
gtaaagccca gaggccaagg aagtttccag gacagccgga 660 cagctcccgc
agcaacctgg ttccagcagc atcggctgct ggctgctctc ccagcactgg 720
ggttcggggg gaggggggtg gccaaagggg cgtttcctct gcttttggtg tttgtacatg
780 taaaagattg acctgt 796 36 214 PRT Eukaryote 36 Met Thr Leu Ala
Ala Tyr Lys Glu Lys Met Lys Glu Leu Pro Leu Val 1 5 10 15 Ser Leu
Phe Cys Ser Cys Phe Leu Ser Asp Pro Leu Asn Lys Ser Ser 20 25 30
Tyr Lys Tyr Glu Gly Trp Cys Gly Arg Gln Cys Arg Arg Lys Gly Gln 35
40 45 Ser Gln Arg Lys Gly Ser Ala Asp Trp Arg Glu Arg Arg Glu Gln
Ala 50 55 60 Asp Thr Val Asp Leu Asn Trp Cys Val Ile Ser Asp Met
Glu Val Ile 65 70 75 80 Glu Leu Asn Lys Cys Thr Ser Gly Gln Ser Phe
Glu Val Ile Leu Lys 85 90 95 Pro Pro Ser Phe Asp Gly Val Pro Glu
Phe Asn Ala Ser Leu Pro Arg 100 105 110 Arg Arg Asp Pro Ser Leu Glu
Glu Ile Gln Lys Lys Leu Glu Ala Ala 115 120 125 Glu Glu Arg Arg Lys
Tyr Gln Glu Ala Glu Leu Leu Lys His Leu Ala 130 135 140 Glu Lys Arg
Glu His Glu Arg Glu Val Ile Gln Lys Ala Ile Glu Glu 145 150 155 160
Asn Asn Asn Phe Ile Lys Met Ala Lys Glu Lys Leu Ala Gln Lys Met 165
170 175 Glu Ser Asn Lys Glu Asn Arg Glu Ala His Leu Ala Ala Met Leu
Glu 180 185 190 Arg Leu Gln Glu Lys Asp Lys His Ala Glu Glu Val Arg
Lys Asn Lys 195 200 205 Glu Leu Lys Glu Glu Ala 210 37 3976 DNA
Eukaryote CDS (51)...(1790) 37 cggcgatggc ggcggctgct gtggtggcag
cgacggtccc cgcgcagtcg atg ggc 56 Met Gly 1 gcg gac ggc gcg tcc tcc
gtg cac tgg ttc cgc aaa gga cta cgg ctc 104 Ala Asp Gly Ala Ser Ser
Val His Trp Phe Arg Lys Gly Leu Arg Leu 5 10 15 cac gac aac ccc gcg
ctg tta gct gcc gtg cgc ggg gcg cgc tgt gtg 152 His Asp Asn Pro Ala
Leu Leu Ala Ala Val Arg Gly Ala Arg Cys Val 20 25 30 cgc tgc gtc
tac atc ctc gac ccg tgg ttc gcg gcc tcc tcg tca gtg 200 Arg Cys Val
Tyr Ile Leu Asp Pro Trp Phe Ala Ala Ser Ser Ser Val 35 40 45 50 ggc
atc aac cga tgg agg ttc cta ctg cag tct cta gaa gat ctg gac 248 Gly
Ile Asn Arg Trp Arg Phe Leu Leu Gln Ser Leu Glu Asp Leu Asp 55 60
65 aca agc tta aga aag ctg aat tcc cgt ctg ttt gta gtc cgg ggt cag
296 Thr Ser Leu Arg Lys Leu Asn Ser Arg Leu Phe Val Val Arg Gly Gln
70 75 80 cca gct gat gtg ttc cca agg ctt ttc aag gaa tgg ggg gtg
acc cgc 344 Pro Ala Asp Val Phe Pro Arg Leu Phe Lys Glu Trp Gly Val
Thr Arg 85 90 95 ttg acc ttt gaa tat gac tcc gaa ccc ttt ggg aaa
gaa cgg gat gca 392 Leu Thr Phe Glu Tyr Asp Ser Glu Pro Phe Gly Lys
Glu Arg Asp Ala 100 105 110 gcc att atg aag atg gcc aag gag gcg ggt
gtg gag gtg gtg act gag 440 Ala Ile Met Lys Met Ala Lys Glu Ala Gly
Val Glu Val Val Thr Glu 115 120 125 130 aac tct cac acc ctt tat gac
tta gac aga atc atc gaa ctg aat ggg 488 Asn Ser His Thr Leu Tyr Asp
Leu Asp Arg Ile Ile Glu Leu Asn Gly 135 140 145 cag aaa cca ccc ctt
acc tac aag cgc ttt cag gct ctc atc agc cgt 536 Gln Lys Pro Pro Leu
Thr Tyr Lys Arg Phe Gln Ala Leu Ile Ser Arg 150 155 160 atg gag ctg
ccc aag aag cca gtg ggg gct gtg agc agc cag cat atg 584 Met Glu Leu
Pro Lys Lys Pro Val Gly Ala Val Ser Ser Gln His Met 165 170 175 gag
aac tgc aga gct gag atc cag gag aac cat gat gac acc tat ggc 632 Glu
Asn Cys Arg Ala Glu Ile Gln Glu Asn His Asp Asp Thr Tyr Gly 180 185
190 gtg cct tcc tta gag gaa ctg gga ttc ccc aca gaa gga ctt ggc cca
680 Val Pro Ser Leu Glu Glu Leu Gly Phe Pro Thr Glu Gly Leu Gly Pro
195 200 205 210 gct gtt tgg caa gga gga gag aca gaa gct ctg gcc cgc
ctg gat aag 728 Ala Val Trp Gln Gly Gly Glu Thr Glu Ala Leu Ala Arg
Leu Asp Lys 215 220 225 cac ttg gaa cgg aag gcc tgg gtt gcc aac tat
gag aga cct cgg atg 776 His Leu Glu Arg Lys Ala Trp Val Ala Asn Tyr
Glu Arg Pro Arg Met 230 235 240 aat gcc aat tcc ttg ctg gcc agc ccc
aca ggc ctc agc ccc tac ctg 824 Asn Ala Asn Ser Leu Leu Ala Ser Pro
Thr Gly Leu Ser Pro Tyr Leu 245 250 255 cgc ttt ggc tgc ctc tcc tgc
cgc ctc ttc tac tac cgc ctg tgg gac 872 Arg Phe Gly Cys Leu Ser Cys
Arg Leu Phe Tyr Tyr Arg Leu Trp Asp 260 265 270 ttg tac aga aag gtg
aag agg aac agc aca ccc ccc ctc tcc tta ttt 920 Leu Tyr Arg Lys Val
Lys Arg Asn Ser Thr Pro Pro Leu Ser Leu Phe 275 280 285 290 gga caa
ctc cta tgg cga gaa ttc ttc tat aca gcg gcc acc aac aac 968 Gly Gln
Leu Leu Trp Arg Glu Phe Phe Tyr Thr Ala Ala Thr Asn Asn 295 300 305
ccc agg ttt gac cga atg gag ggg aac ccc atc tgc atc cag atc ccc
1016 Pro Arg Phe Asp Arg Met Glu Gly Asn Pro Ile Cys Ile Gln Ile
Pro 310 315 320 tgg gac cgc aac ccc gaa gcc ctg gcc aag tgg gcc gag
ggc aag aca 1064 Trp Asp Arg Asn Pro Glu Ala Leu Ala Lys Trp Ala
Glu Gly Lys Thr 325 330 335 ggc ttc cct tgg att gac gcc atc atg acc
caa ctg agg cag gag ggc 1112 Gly Phe Pro Trp Ile Asp Ala Ile Met
Thr Gln Leu Arg Gln Glu Gly 340 345 350 tgg atc cac cac ctg gcc cgg
cac gct gtg gcc tgc ttc ctc acc cga 1160 Trp Ile His His Leu Ala
Arg His Ala Val Ala Cys Phe Leu Thr Arg 355 360 365 370 ggg gac ctc
tgg gtc agc tgg gag agc ggg gtc cgg gta ttt gat gag 1208 Gly Asp
Leu Trp Val Ser Trp Glu Ser Gly Val Arg Val Phe Asp Glu 375 380 385
ttg ctc ctg gat gca gat ttc agc gtg aat gca ggc agc tgg atg tgg
1256 Leu Leu Leu Asp Ala Asp Phe Ser Val Asn Ala Gly Ser Trp Met
Trp 390 395 400 ctg tcc tgc agt gct ttc ttc caa cag ttc ttc cac tgc
tac tgc cct 1304 Leu Ser Cys Ser Ala Phe Phe Gln Gln Phe Phe His
Cys Tyr Cys Pro 405 410 415 gtg ggc ttt ggc cga cgc acg gac ccc agt
ggg gac tac atc cgg cga 1352 Val Gly Phe Gly Arg Arg Thr Asp Pro
Ser Gly Asp Tyr Ile Arg Arg 420 425 430 tac ctg ccc aaa ctg aaa ggc
ttc ccc tct cga tat atc tat gag ccc 1400 Tyr Leu Pro Lys Leu Lys
Gly Phe Pro Ser Arg Tyr Ile Tyr Glu Pro 435 440 445 450 tgg aat gct
ccc gag tcg gtt cag aag gcc gct aag tgc atc att ggc 1448 Trp Asn
Ala Pro Glu Ser Val Gln Lys Ala Ala Lys Cys Ile Ile Gly 455 460 465
gtg gac tac cca cgg ccc atc gtc aac cac gca gag act agt cgg ctc
1496 Val Asp Tyr Pro Arg Pro Ile Val Asn His Ala Glu Thr Ser Arg
Leu 470 475 480 aac att gag cgg atg aag cag atc tac caa cag ctg tca
cga tac cgg 1544 Asn Ile Glu Arg Met Lys Gln Ile Tyr Gln Gln Leu
Ser Arg Tyr Arg 485 490 495 ggg ctc tgt ctg ttg gca tct gtc cct tcc
tgt gta gaa gac ctc agt 1592 Gly Leu Cys Leu Leu Ala Ser Val Pro
Ser Cys Val Glu Asp Leu Ser 500 505 510 cac cct gtg gca gag cct ggt
tct agc cag gct ggg agc atc agc aac 1640 His Pro Val Ala Glu Pro
Gly Ser Ser Gln Ala Gly Ser Ile Ser Asn 515 520 525 530 aca ggc ccc
aga cca ctg tcc agt ggc cca gcc tcc ccc aaa cgc aag 1688 Thr Gly
Pro Arg Pro Leu Ser Ser Gly Pro Ala Ser Pro Lys Arg Lys 535 540 545
ctg gaa gca gct gag gaa cct cca ggt gaa gaa ctg agc aag cgg gct
1736 Leu Glu Ala Ala Glu Glu Pro Pro Gly Glu Glu Leu Ser Lys Arg
Ala 550 555 560 aga gtg aca gtg act cag atg cct gcc cag gag cca cca
agc aag gac 1784 Arg Val Thr Val Thr Gln Met Pro Ala Gln Glu Pro
Pro Ser Lys Asp 565 570 575 tcc tga gactggagag ccattgctcc
gtgagcaaag cccaggtgcc tgagctgcca 1840 Ser * tggccacaga gaagacatgg
aacctacaga gaagacagtc accaacagac agagcgagcg 1900 actgtgtgtg
tgcagaggga ggtgtggtgt gccgtttgcg tgtgcatgca tctgtttaca 1960
ctctcatgat cctgaatgtt gcctgtgctg gaggagcccc tagatcatgc cttcttacca
2020 gggctgtttc ttgacttcca gacataagac tagaacccgc agcagtaacc
gtcagcccaa 2080 atctgcccct gggagcccca atagggtggt aagaccctag
cttgaattct ggtctctgcc 2140 tccccagact cttcttcctc cctcctttta
acaaggagct ggagggccac atttttgact 2200 ctcatctaaa gcatggagtt
tcagaggcag tcagagtcct gctgacttag ttcccacttt 2260 tctgacacta
gaacctgagc aggctggaat agatgtgtcc tgttgatctt aaacagcctg 2320
gccagtcttc ttataaaatc ctgtgccatt aacaggcttc cctgatgtct aaggctacag
2380 actagtgtgt tgtgtgccca gtactgctta tgtcagcctc agacataata
tcagtctttg 2440 tagaaccttc taaaaaaaac cacatgggga atagactccc
agtcttctgt cccttcccta 2500 gcagctaagg tccagtctcg accttctaga
agctgtggac aggctagggt ctgaactggt 2560 gaaagaaacc caggtcccac
agctgcaggg cccctggttc ctctggctgt actcctgaca 2620 ccacatgctc
cagccagtac tgctgatatc cagccaggca agctggacag cctggctggt 2680
cagcacctgc cctgcagtgt cagctgccca ggactgagct tccggagact cagacagact
2740 taggggtgga gcactgcctc tggcagttgg cgagaggtca gagaccatgc
ctggcacatc 2800 aacatcttcg cagagcagca gtgaaggatt gacatagaga
agtcaagcct tgctttccag 2860 gggagccaac tctccctccc actgttgggt
catatggaga aagaagttat gaaaggatct 2920 gggggtacct gagcaagtct
tccttccacc ccgtggcctg catttgagcc acagtgtgtg 2980 tgtgtgtgtg
tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtgtg tgtgtgtaga 3040
gagagagaga gagagagaga gagagagaga gagagagaga gagagagaga gagagtttgt
3100 ttctgtttgg atttttgttc tcacatgtaa cattaagctg gcctctgggc
cttttcctct 3160 ctacctcccc tgtgaccttt cctagcctca gagttgttaa
tgcccttggc cctggccttt 3220 tttttgtgtc agaccagaac cctggggtca
ggctcccccc tccagctgtc tagcacatct 3280 gacaggcttc tttttgagat
ggcctcaggt tttctcagca gagagctgcc tttagtccaa 3340 ctgtttatgt
tcatcatcct gactagaagc atcctacgat tgtgtgaaga aacggcatct 3400
gtgatgccat gttcagagtc atggggtgtg gcctccctgt
ccctagcccc aggccaagag 3460 gaaagggcca aaggctcttg ctggagggac
agtagaatgc gtctggagaa ctggtcccag 3520 aggagcaaag gcttattctg
gggccagtat ttattttgca acatcttcag ctatggggac 3580 aatggccttc
tctgcttttt tgatgatggc tctctcctca aggtacaagt tggcaaggtc 3640
atctgtcctt ccacctcctt gacatgttgg cccatttcca ggacagcctt ccagtgaatg
3700 gagcagacta ttccacagct gtgggataga gtgtcttgga gccctggaat
gacttcatgc 3760 ctccttttgc ctagcctgag tggccctgag gactgtcaca
gaacagtgcc ccatgtcctg 3820 ctcctgggcc cgagcatggg gaagagatgg
ttgcaggcaa gagcacttta cagcattccc 3880 cattgctggg aaggttgttt
ctcctacagt gtgtgaatac ttacctgttt tataaatgtc 3940 tgatcctgtc
tgagtaaaaa aaaaaaaaaa aaaaaa 3976 38 579 PRT Eukaryote 38 Met Gly
Ala Asp Gly Ala Ser Ser Val His Trp Phe Arg Lys Gly Leu 1 5 10 15
Arg Leu His Asp Asn Pro Ala Leu Leu Ala Ala Val Arg Gly Ala Arg 20
25 30 Cys Val Arg Cys Val Tyr Ile Leu Asp Pro Trp Phe Ala Ala Ser
Ser 35 40 45 Ser Val Gly Ile Asn Arg Trp Arg Phe Leu Leu Gln Ser
Leu Glu Asp 50 55 60 Leu Asp Thr Ser Leu Arg Lys Leu Asn Ser Arg
Leu Phe Val Val Arg 65 70 75 80 Gly Gln Pro Ala Asp Val Phe Pro Arg
Leu Phe Lys Glu Trp Gly Val 85 90 95 Thr Arg Leu Thr Phe Glu Tyr
Asp Ser Glu Pro Phe Gly Lys Glu Arg 100 105 110 Asp Ala Ala Ile Met
Lys Met Ala Lys Glu Ala Gly Val Glu Val Val 115 120 125 Thr Glu Asn
Ser His Thr Leu Tyr Asp Leu Asp Arg Ile Ile Glu Leu 130 135 140 Asn
Gly Gln Lys Pro Pro Leu Thr Tyr Lys Arg Phe Gln Ala Leu Ile 145 150
155 160 Ser Arg Met Glu Leu Pro Lys Lys Pro Val Gly Ala Val Ser Ser
Gln 165 170 175 His Met Glu Asn Cys Arg Ala Glu Ile Gln Glu Asn His
Asp Asp Thr 180 185 190 Tyr Gly Val Pro Ser Leu Glu Glu Leu Gly Phe
Pro Thr Glu Gly Leu 195 200 205 Gly Pro Ala Val Trp Gln Gly Gly Glu
Thr Glu Ala Leu Ala Arg Leu 210 215 220 Asp Lys His Leu Glu Arg Lys
Ala Trp Val Ala Asn Tyr Glu Arg Pro 225 230 235 240 Arg Met Asn Ala
Asn Ser Leu Leu Ala Ser Pro Thr Gly Leu Ser Pro 245 250 255 Tyr Leu
Arg Phe Gly Cys Leu Ser Cys Arg Leu Phe Tyr Tyr Arg Leu 260 265 270
Trp Asp Leu Tyr Arg Lys Val Lys Arg Asn Ser Thr Pro Pro Leu Ser 275
280 285 Leu Phe Gly Gln Leu Leu Trp Arg Glu Phe Phe Tyr Thr Ala Ala
Thr 290 295 300 Asn Asn Pro Arg Phe Asp Arg Met Glu Gly Asn Pro Ile
Cys Ile Gln 305 310 315 320 Ile Pro Trp Asp Arg Asn Pro Glu Ala Leu
Ala Lys Trp Ala Glu Gly 325 330 335 Lys Thr Gly Phe Pro Trp Ile Asp
Ala Ile Met Thr Gln Leu Arg Gln 340 345 350 Glu Gly Trp Ile His His
Leu Ala Arg His Ala Val Ala Cys Phe Leu 355 360 365 Thr Arg Gly Asp
Leu Trp Val Ser Trp Glu Ser Gly Val Arg Val Phe 370 375 380 Asp Glu
Leu Leu Leu Asp Ala Asp Phe Ser Val Asn Ala Gly Ser Trp 385 390 395
400 Met Trp Leu Ser Cys Ser Ala Phe Phe Gln Gln Phe Phe His Cys Tyr
405 410 415 Cys Pro Val Gly Phe Gly Arg Arg Thr Asp Pro Ser Gly Asp
Tyr Ile 420 425 430 Arg Arg Tyr Leu Pro Lys Leu Lys Gly Phe Pro Ser
Arg Tyr Ile Tyr 435 440 445 Glu Pro Trp Asn Ala Pro Glu Ser Val Gln
Lys Ala Ala Lys Cys Ile 450 455 460 Ile Gly Val Asp Tyr Pro Arg Pro
Ile Val Asn His Ala Glu Thr Ser 465 470 475 480 Arg Leu Asn Ile Glu
Arg Met Lys Gln Ile Tyr Gln Gln Leu Ser Arg 485 490 495 Tyr Arg Gly
Leu Cys Leu Leu Ala Ser Val Pro Ser Cys Val Glu Asp 500 505 510 Leu
Ser His Pro Val Ala Glu Pro Gly Ser Ser Gln Ala Gly Ser Ile 515 520
525 Ser Asn Thr Gly Pro Arg Pro Leu Ser Ser Gly Pro Ala Ser Pro Lys
530 535 540 Arg Lys Leu Glu Ala Ala Glu Glu Pro Pro Gly Glu Glu Leu
Ser Lys 545 550 555 560 Arg Ala Arg Val Thr Val Thr Gln Met Pro Ala
Gln Glu Pro Pro Ser 565 570 575 Lys Asp Ser 39 629 DNA Eukaryote
misc_feature (1)...(629) r = G or A y = C or T n = A,T,C or G 39
ttggcacaca agtctgtctt caggacagct gatccatttt acttacraat tcagaaagta
60 aacattggca gtatggatct ggttacttca tggtaactgc tctagaattt
acgccaaggc 120 catctctttt gcctcactgt ttagtgaccg gagtaaagca
tggggccact gaaactccac 180 tttacaattg ggcttctaaa tttaaggaaa
aattttttga tttaaccaca actggattcc 240 aaagttcatc ttattcyaaa
ttaggcccac tgagcctgtg atgttttgga atatatgatt 300 agtccacttg
gttcactgga tgttacctat catgttatgt agagaaacag ccataactat 360
tggtcacgat gtcgtcctcc gaattgggaa tggctctgtt gttggaaaca aagtatttgt
420 aaacacgttg atcaaagcgg tgtgctttgg cctttccggg aatcactgat
tatgtttgaa 480 aacttccttt aattgtattt gcaataagct attntccctt
ntnatgncnc tgccatgctt 540 ccttgctttg cactgtggtc gcatgccatc
ngctggttaa cccangatgg cttgctgcnc 600 tgatatncac catgcnaaat
accacttct 629 40 2461 DNA Eukaryote 40 tgaattgcag taactagcct
tgcctttcta ttctgtagaa atgacagggt cttcacaatc 60 cttcaccagt
ggctactaag ctataattag ctgaatagaa agaatgtgga agtggtctga 120
ggcatataga gcatatgcca agaacactac catatatggc atcagctttg gttaccagag
180 aaattttctt agtcattaga ccatataaca gtaatatatc atatgtaaat
ctttagattt 240 caatttgaga atcctccaaa aaaaaggagc aaagaatgca
taagctatgt gttggcaaaa 300 gtaatttata ttaaaatttt gacctgcctt
tgtaagatta agtggtaaat gtcatagtgg 360 tgggttttta cgtcttaacc
aatctctgag gtttatttct cctgcagggg atggttcatg 420 gcctctcttc
ccgctgtagg aagatagcag aaggatgagg attaattgta gcatttcact 480
gatcctcgtc ccagggacta gggacaatag aaatctgcaa acatggagag tctgtcataa
540 atatttgctt tttgaaggtg ttggtctttg ttgatttctg tcagaaaatg
gcattataca 600 aattatgggg agcaaccaac ttttctgttc tgtttttgaa
gtgctactat gaaccattca 660 gagtcgtatt tttttttttt aaaattttgg
ccagatatcc ccagctaatg aaaaatagtc 720 accattcctt gaaaaagttg
gaagctagaa cccccaattc caaattattg ttgaagatgt 780 ttctcaggct
actgtatata gaaataatgt ttttaagaaa aatcaaagag aggagaaaaa 840
aaaaacctat gcagagaccc tactactttg tggtttctat tgtccctata catcatttca
900 gcaaatctac tggcagttct tgtcagcaag tccttcagtg catatgctgc
acaaaacaaa 960 acaaaaatct gcatggcacc aaaaaccaaa caagcaaacc
aaaaacccag acaccctatg 1020 tatctgttgg aggcatgtag gtggtacaaa
tgactagcca tgagcacaca tggcttcttg 1080 tcatgtcact tttcataatt
atttactgca aaatgattga gaggcttttg gtgcaggcag 1140 ccattagcct
gcttcctttg ttacctctgg atcactttgc agtaaattgc aggtctttta 1200
aaagattcaa gcttcggttt tctcaaaaca aaacaattat cctgtcttac ctgaaaatgc
1260 agggttgtgg gcaaaagagg ctggttataa taatgccctc atattgagtg
gtctgtaaat 1320 ggctgcacac ttcaggcact agagttgccg aggatgcgtt
gttaatgtga ccttgactgg 1380 ctttacaggg gtgtagaaca gtctacacgg
gcgactattt gcatccatct tgctctcgag 1440 gtggatggaa ataagaaaag
gctggagtgt gtaagtcatg cacataagta ttcactgtaa 1500 attttatttt
catttttaac ccaattatgg tactttgtcc aatgcacaac tgatctctca 1560
gtagatattc atttgaaaat agtgtggcct tgaccagcga gaaggggaag aagtgactta
1620 gcttgtgtta agatgacctg tttgctgaga gtggtcattc tgcagcaccc
taatgtcatg 1680 gttttgatta gggagagtta atgtttttga ccctgaattg
agttttcttc tatttttagg 1740 aagtatcaga attgctctga tgagtaacaa
agttgactgt tttgatgtcc aatctcaggt 1800 tttaaaatag agtggtataa
aagtccactg ttactaattc ttaagacaat tttgatttag 1860 tgtgccctaa
aagtcacgtg cataataagg cctgctcaga gggcagggcc tccatctgtt 1920
tgctcctttc catgttgtac gcacttcact tgaaaaggtg tcaagtgact ttgcattgta
1980 gatttccatt ttaaccccaa catagttctc aaagataaag cactttttga
acatgaaata 2040 catgggtaat gtgtgatgtg gatcatggtt tctcaggccc
ctagataatc cacttctgag 2100 tattgttcta tgtaaggaga atagaggtct
tcgctaatgt tcgagtttgt attcctgaat 2160 ggaatgcact tgctagtttc
caatggatgg gagagtaaac actgctgcat tcacaattga 2220 tacgttgctt
tcccttgagc cttaaggtaa cttttctttt ctgtcaacaa cagcactgaa 2280
gttctagtaa gtgaatgaga ttatctgttt tcagggttgg ttttagagta ctgtaaatta
2340 attagctgtc ttcctaaaga ggaactccct ttaactccct tcgatagact
gaaagtgggt 2400 gtggggaggg ggagggaaga gagggaggta gtttgtagaa
aaaaaaaaaa aaaaaaaaaa 2460 a 2461 41 1131 DNA Eukaryote
misc_feature (1)...(1131) r = G or A n = A,T,C or G 41 ggccccccct
anaaggtcga ggntatcgat aagcttnaat atcgaattcg gcacgaggcc 60
accaggtctt tgcattgtct ctttaaaagt ggtgtataag ggggaaattg gcaagacaga
120 catttctaaa cagaggggaa cacagacaga cagacagaca gacagacaca
caaacacaca 180 aacacacaca cacacacaca cacacacaca cacacacaca
cacacacaca cacacacaca 240 cacaccccag actgcgtatg tggcataaca
tacagcttgc atgggaagca gccccctgcr 300 cattgcttat acatcctcga
gtcctttcat cttttttcct aaaacgtgtg cacccgctat 360 aaagtgggtg
atgggctcgt cagagctggg ctgattctgt ggccggtgac caccatgcct 420
caggtccctc aacctccatc acccatggcc caatccataa ctgccaccct tgaaaaccca
480 aagcagtctg agggtgctct ctgcctgtca ctcagaggcc tgggacgttg
aacccaaaaa 540 agctaaactt atgaaagccg ggctgaaatg gggcccgggg
cctgggatag ctcaggcagg 600 ggttttccac tctgatgttt ccactgggcc
agttttgttt ctttgtctct attttctctg 660 ttcatcccgc tgagtgtttg
tatccatgat gattccagca tgaagtacgt agcacactcc 720 agttaggaga
aattttttaa agatacaaga ctagcgtggt ggtgagatga gatagtcttc 780
tcgtgctcgc agcaacctga aggggcaata aggacaaaga aggccatgtg gcagggttag
840 ccccctccag accaggggta caacggacag ttgtggtgag cctcggaaag
gcaggggtaa 900 ccttccctct ccgttcttca cccatggcca gagcaaggca
ggtagtgaaa gggatatgct 960 tgatgcagaa aagccagctc aggcatggca
ggtgggattt atagctggtt ttgtttaaag 1020 cgaaggcctg atatttgata
aatgcagtaa ccagcggttg agagtgacaa gcccttaaat 1080 gcgaacatta
atcaaaggag aacttaaacg gcccccttta cagaaggact t 1131 42 1473 DNA
Eukaryote 42 cgagtttttt ttttttatgt actttgaaaa tatatttaaa aacattaaaa
attctatatt 60 taaaacatat attatatgtt aattggtaca cttaaataga
acctgtattt acaataggct 120 tctgatgtgg ttaagtttta atgccaattt
ttttttcaat aacataatta tataaatata 180 ctaaaataca ataaatattt
ttcttgtttt acatggtgaa taatatcttt accatagaga 240 gaacaaggcc
acagacattt acttacagtt tcaatgggaa tcactataaa aagcatcagg 300
cctgctgcca tgcatgaaac acttctgcca aaaagagacc acagcaagac tttcagaaca
360 gaacagaaca gaacaggacg gaaacagaac gaacagaaac agaggagaga
ttttaacaaa 420 tcaatctcag gtcaacataa accaccgaca tggagctatg
atgtatctta gtgggtatga 480 gagccagcca ctgaccacac agttgcggag
ggtctcctat gaagccacct aatcgacctg 540 gcccttcgaa taccgtgaga
ttgtgatggg gctcctttta tttgtttgac taacgtctct 600 cagaatgaag
ctgcaaaaag ttagcatata gcagatattc aaagcattcc ttaataggtt 660
aaaaatgatg acagagatta atgttgtcaa acggcacaaa acaatctagg ctacgtgaag
720 tcttccaaaa acaggggatt cagtgggact ccagaagaca gactagttct
aaaggaacag 780 ttgaacaaaa agaaactatt tgctgatggt atcttcactc
cctgagtcac agtggacagc 840 cactttgttt caccctttcc actcctaaga
tgaagcaatt gtttgcctct ttttctgatg 900 cccaggagcc cagtcaggta
accactaaca cattcgcgct ggcggaaaac ctcactaggg 960 aaatgggctt
aacactagtt ctcattgggg ccattcattc aggcttccag cttgacttct 1020
cctaacccca agaggtaaag tgtagaaggg acccttgtgc tgaatggaca gaactatcag
1080 gagctttctg tgctcttcac ttaagcagta tttcctcctg tgttcttgtc
tctttcacag 1140 tgaaagcacc ttcctatgcc ttgtcattct agcccttaca
gacagacatt gctcattctg 1200 cctaagtttt ggtgcttttt ctggttttgt
ttgtttgttt tcttctttct tttttccttt 1260 caccaaaatg tctcaaaaaa
ataaataaat aaaacctagg cttcctgaag tctaagcgca 1320 aagaaagtta
agtctcttca cagcaaacat ttcccatcat gctgcactga tagcatcact 1380
gctatgccat atttggatcc aaagctgctc caggttaatc caactttatc cataattatt
1440 taaaatggga tggaggccat aaatggattt gag 1473 43 326 DNA Eukaryote
43 agtctgggac taaaacgtca cagcagaaaa aaaataaaaa aaaataattt
gctttttctt 60 tctttcattt agcagcataa ataagtttgg ccactgggag
tacagtacag gggtgggaca 120 acgatcccgt atttgaagac ctacttctag
caccagcatc aagaactaaa tccacctcag 180 gactcacaga acccaggaca
acttgccatc tttgagcaac atatgcattg aagagtgtat 240 atagaagcaa
cagtaaatag attaacagag gctaatactg tgattgattg acattggcaa 300
tggttggcaa aaaaaaaaaa aaaaaa 326 44 429 DNA Eukaryote misc_feature
(1)...(429) n = A,T,C or G 44 acttgataaa attgtatttt tttttctaca
gtcatttgta caatttgtta caaaaccata 60 gaagactaca acttgtttta
aatcattttt ggtctgcaaa tatgtaaaat ctgtggtgca 120 attatcatgt
atttacaggg ccttgttagt cattttcaat gattatttca acaatgtcac 180
actctcaaca taagacatgg cttaagacaa atatattagt acatanatat tctgagaaca
240 tatttccatn aatggaaagt ngctgctaat acanatacag aatatacata
agntgttttc 300 tagcttttta aaacagtttt taaaatggna angtgaaaaa
agagccccta ggancatttt 360 atcccaaaaa aatccttacn aaatattnaa
ggggccaggg ggggaattaa aaatctaaaa 420 anggtggtc 429 45 1210 DNA
Eukaryote misc_feature (1)...(1210) r = G or A m = A or C s = G or
C w = A or T 45 aargggrcca ccccaccgsg ctaaaggccc aggggccccc
cccttggagm cccaggggtt 60 ttggcccmcc ccctcaccca aatggtctgc
caatgaccca ggtactcaca acatgttcca 120 ggaggagmct ggggccagga
ttttgaccag agggtatggg aagggaaagg ggagaagaaa 180 tcgacattta
tttttattat ttattttaaa tgtttacawt ttctttgtgt tgttccaagc 240
cctgaataga aacagatagc attaaaggac tctgttccca ccccttctct gtctctctct
300 cccccacttg tgctaactta ggataacact ctctatttcg ttttgtttct
aaagtgattt 360 gtggacttgt gccgtgtgaa ctgcattaaa aaggttctgt
tttcaaagat cgattgtcgt 420 tcctgtgggg acagtggctc ctaagaaatc
tgcattgtag gagaagacaa tgaaagaccc 480 tggccctgtc tctcaaaact
taactctctg tatgatttaa aaaaaaattc catttacttt 540 actttgtggt
tacttgattt tgaggaagaa aatattcaac tttgtataaa gactaggtat 600
cagggtttct tttgcagtgg gagttgtata tatatcgtat tttggtatat cgtagaaact
660 caagctttat gcatccgtat ttgggatatg tcaatgacgt gcagtgaaat
ttgctattag 720 accctggagg caaacgagtt gtacaaggtt ttatggctcc
atggggaatt ctaatttcct 780 ttctggggac cttttgtccc gtttttacag
taatggtgaa atggtcctag gagggtctct 840 ctagtcgaat tctccaggca
ggaccacgtg ctcaaaaaat ctttgtatag ttttaaattt 900 ttgaggagta
tctctgctca gaagcatctg tggtggtgtg tgttgcgttg ttctgtgtac 960
tgtgtgtgac acaagcctac agtatttgca ctaaggaaag ctgtttagag cttgctgcta
1020 tggagggaag aacatattaa aacttatttt ccctcggggw ttrtwcwmgt
tttatgtwct 1080 tgttgtcttg ttggctttcc tactttccac tgagtagcat
tttgtagaat aaaatgaatt 1140 aagatcagmw rwrwrmaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1200 aaaaaaaaaa 1210 46 700 DNA
Eukaryote misc_feature (1)...(700) n = A,T,C or G 46 aattccccat
ggagccataa aaccttgtac aactcgtttg cctccagggt ctaatagcaa 60
atttcactgc acgtcattga catatcccaa atacggatgc ataaagcttg agtttctacg
120 atataccaaa atacgatata tatacaactc ccactgcaaa agaaaccctg
atacctagtc 180 tttatacaaa gttgaatatt ttcttcctca aaatcaagta
accacaaagt aaagtaaatg 240 gaattttttt taaatcatac agagagttaa
gttttgagag acagggccag ggtctttcat 300 tgtcttctcc tacaatgcag
atttcttagg agccactgtc cccacaggaa cgacaatcga 360 tctttgaaaa
cagaaccttt ttaatgcagt tcacacggca caagtccaca aatcactttn 420
gaaacaaaac gaaatagaga gtgttatcct aagtnagcac aagtgggggn gagngagaca
480 gagaaggggt gggaacagag tcctttaatg cnatctgttt ctattcaggc
ttggaacaac 540 acaaagaaat gtaaacattt agnataaata atagaataaa
tgtcgggttc ttctcccctg 600 tcccttccca tacccnctgg caaaatctgn
cccaggtcct cccggaacat ggtgngagta 660 cctgggtcca ttgnagncca
tttggngagg gcgtggccaa 700 47 3316 DNA Eukaryote CDS (94)...(993) 47
ggcacgagcc gaggctcagc acagcacgga taggggcgcg gagcgcactg agaaccctac
60 tttcccgtga gcccgagccc ggcaaatggg cga atg aag aag gag agc agg gac
114 Met Lys Lys Glu Ser Arg Asp 1 5 atg gac tgc tat ctg cgt cgc ctc
aaa cag gag ctg atg tcc atg aag 162 Met Asp Cys Tyr Leu Arg Arg Leu
Lys Gln Glu Leu Met Ser Met Lys 10 15 20 gag gtg ggg gat ggc tta
cag gat cag atg aac tgc atg atg ggt gca 210 Glu Val Gly Asp Gly Leu
Gln Asp Gln Met Asn Cys Met Met Gly Ala 25 30 35 ctt caa gaa ctg
aag ctc tta cag gtg cag aca gca ttg gaa cag ctg 258 Leu Gln Glu Leu
Lys Leu Leu Gln Val Gln Thr Ala Leu Glu Gln Leu 40 45 50 55 gag atc
tct gga ggc gcg ccc acc ttc agc tgc cct aag agc tca cag 306 Glu Ile
Ser Gly Gly Ala Pro Thr Phe Ser Cys Pro Lys Ser Ser Gln 60 65 70
gaa cag acc gag tgc cct cgc tgg cag ggt agt gga ggg cct gct ggg 354
Glu Gln Thr Glu Cys Pro Arg Trp Gln Gly Ser Gly Gly Pro Ala Gly 75
80 85 ctt gct gcc tgt ccc tcc tcc agt caa cca tct ttt gac ggc agc
ccc 402 Leu Ala Ala Cys Pro Ser Ser Ser Gln Pro Ser Phe Asp Gly Ser
Pro 90 95 100 aag ttt cca tgc cgt agg agt atc tgt ggg aag gag ctg
gct gtc ctt 450 Lys Phe Pro Cys Arg Arg Ser Ile Cys Gly Lys Glu Leu
Ala Val Leu 105 110 115 ccc aag acc cag atg cca gag gac cag agc tgt
acc caa caa ggg ata 498 Pro Lys Thr Gln Met Pro Glu Asp Gln Ser Cys
Thr Gln Gln Gly Ile 120 125 130 135 gag tgg gtg gag cca gat gac tgg
acc tcc acg ttg atg tca cgg ggc 546 Glu Trp Val Glu Pro Asp Asp Trp
Thr Ser Thr Leu Met Ser Arg Gly 140 145 150 aga aat cgg cag cct ctg
gtg ttg gga gac aat gtt ttc gca gac ctg 594 Arg Asn Arg Gln Pro Leu
Val Leu Gly Asp Asn Val Phe Ala Asp Leu 155 160 165 gtg ggc aac
tgg cta gac tta cca gaa ctg gaa aag ggc ggg gag agg 642 Val Gly Asn
Trp Leu Asp Leu Pro Glu Leu Glu Lys Gly Gly Glu Arg 170 175 180 ggt
gag act ggg gga tcc ggt gaa ccc aaa gga gaa aaa ggt cag tcc 690 Gly
Glu Thr Gly Gly Ser Gly Glu Pro Lys Gly Glu Lys Gly Gln Ser 185 190
195 aga gag ctg ggt cgt aag ttt gcc cta act gca aac att ttt agg aag
738 Arg Glu Leu Gly Arg Lys Phe Ala Leu Thr Ala Asn Ile Phe Arg Lys
200 205 210 215 ttc ttg cgt agt gtg cgg cct gac cga gac cgg ctg ctc
aag gag aag 786 Phe Leu Arg Ser Val Arg Pro Asp Arg Asp Arg Leu Leu
Lys Glu Lys 220 225 230 cct ggt tgg atg act cct atg gtt tct gag tca
cga gca gga cgc tcg 834 Pro Gly Trp Met Thr Pro Met Val Ser Glu Ser
Arg Ala Gly Arg Ser 235 240 245 aag aaa gtc aag aag agg agc ctt tct
aag ggc tcg gga cgg ttc cct 882 Lys Lys Val Lys Lys Arg Ser Leu Ser
Lys Gly Ser Gly Arg Phe Pro 250 255 260 ttt tcc agc aca gga gag ccc
aga cat att gaa acc ccg gcc aca agc 930 Phe Ser Ser Thr Gly Glu Pro
Arg His Ile Glu Thr Pro Ala Thr Ser 265 270 275 agt ccc aag gct tta
gaa ccc tcc tgt agg ggc ttt gac att aac aca 978 Ser Pro Lys Ala Leu
Glu Pro Ser Cys Arg Gly Phe Asp Ile Asn Thr 280 285 290 295 gct gtt
tgg gtc tga attcgagaga tgctcactga cctaaaatgc agacttgtga 1033 Ala
Val Trp Val * gggccctggg ggagggtggg cagatggcat ggtcttcagg
ccagatgcaa gttcccatcc 1093 tcagaaagaa agcagagttc ttagtcaggc
ctcagtagaa cagtggagag aggctgtcac 1153 aggccaggct gagctgagtc
cctggagaga atgtgtgtat ttgtgtgtgt gtgtgtgtgt 1213 gtgtgtgtgt
gtgtgtgtgt gtgtgtgtat gtgtgtgtgt atgcgtgtgc atgcactgtt 1273
gttgttagag gctggatgtg acaataattg ggagaggcag gaaaggagtc caggacaagc
1333 ctatgatatt cctccattac cttacccaag acctcatttg aacattctat
atgcaaaggg 1393 gcatttagcc ctcaggtttc ccagaggaac tcccaataaa
gacctgtctc agggaccccc 1453 aaccattttt taatggtctg cttccctgac
aaggcactga tgcaggcaag gggtttgttt 1513 ttgttttaag ggttggtatc
ccagaatgga gcaccggaaa taggaaaatc cctatttata 1573 gcccttccta
ggaccaagat ttcacccatg gctgggtgct ggggacgcag aacaagcaga 1633
ggggtgtgcg tgcgtgcgtg cgtgcgtgcg tgcatgtggt gttgaggaag cctgagatgc
1693 tcccagatct ctaaagtgca gaggagaagc aatgtgcgtt caccccggtg
attccataag 1753 cagccatctc tgagagcaca ctcggctgcc aggaggaaaa
acaggtcagg ccaatctcat 1813 ggttatcaat ggaccctaga gtcatacgct
gcctggtcca gcagtgagag cccatcctga 1873 ctccctgttg cctatcttaa
tgctcctgca gggcagcaga tggttggggt gaacccagag 1933 ataataccca
tacattgaga acatttctta gtctacatct catagtcatt cagcgaactg 1993
gacacatcta cccgcatcac cctggaggtc aacaggggac cctgagggtg gggctgatgc
2053 caggcacttt atatagtgag caggcgtgca agtctgggac ccagggaatc
catctcagcc 2113 cccacccctt agccaggaga gaacaaagta ggcccctgtt
caagcccagc tcggaggctg 2173 ccttagctcc tccttcgccc cctcctgcag
acccagctca gcttgatgag gtgtgacaac 2233 tgcaattaga ggcaagccgc
ctgctgcccc cagagcatta agagcaaatt agagaagaaa 2293 aatcacaaga
gaagctcttc tgcctgcagt ctagactccc aggggactgg gtggaggaag 2353
gaagagctta gggcataggg atgaggaggt aaaagtaaca gcaggaaggg tcacctgcaa
2413 gttcccacgc agttaaatga taggtggcct tttttttttt tttttaatct
gtagcttttt 2473 gtcaggcaat gtgcctatct ctttcagaac aattaatcag
tggggtcaaa gggccctgcc 2533 atgctggctg cccccatcag gctactcaaa
aaggaaagca gttccaagct ccagcctgtg 2593 ggcatcaggc ctatctgctc
tggcctggtg tttatcagct aggctcgctc tttctggtca 2653 aatgggtcct
catccattct gtccccactg aacttctgtc tctggtgaag gaaggtaact 2713
gtagctgcct ctgatggctg ctgcaatgtg tgtggagaat gaacatgtga aaaccccaca
2773 ccctgaaggg tggcacatat gacacattta ctcaagagga cacaggactg
ggacggtgta 2833 ggaagccaac tcatttgttt tgtggactag tcactgttca
cattatttaa atcgactgac 2893 gtgacagact ccttctttga ctgggcactg
tgacagaagg agagaactca gcaatgggaa 2953 agctggcctc cacagctacc
aaggcacaca aagaaatcca gttaaccacc acctggccag 3013 aaaagggtca
agggaccaaa acaaaatgat tagcaagtaa ttttggcttc taagagaacc 3073
cacaggtgtc tgtcaccttg atctttattt ttctgctaca cccaggaaat ggttgctcat
3133 tttacccagt agactcggag aagttaatgc tttcaaggtc acacagtaca
aagctgggat 3193 tgaaacagtt tgtaactgac ttccaatctt gtgttcatgc
tacctggcaa actgtccata 3253 tttgctccac agccagatcc agaataacat
ttgtctcctc tcgtgcaaaa aaaaaaaaaa 3313 aaa 3316 48 299 PRT Eukaryote
48 Met Lys Lys Glu Ser Arg Asp Met Asp Cys Tyr Leu Arg Arg Leu Lys
1 5 10 15 Gln Glu Leu Met Ser Met Lys Glu Val Gly Asp Gly Leu Gln
Asp Gln 20 25 30 Met Asn Cys Met Met Gly Ala Leu Gln Glu Leu Lys
Leu Leu Gln Val 35 40 45 Gln Thr Ala Leu Glu Gln Leu Glu Ile Ser
Gly Gly Ala Pro Thr Phe 50 55 60 Ser Cys Pro Lys Ser Ser Gln Glu
Gln Thr Glu Cys Pro Arg Trp Gln 65 70 75 80 Gly Ser Gly Gly Pro Ala
Gly Leu Ala Ala Cys Pro Ser Ser Ser Gln 85 90 95 Pro Ser Phe Asp
Gly Ser Pro Lys Phe Pro Cys Arg Arg Ser Ile Cys 100 105 110 Gly Lys
Glu Leu Ala Val Leu Pro Lys Thr Gln Met Pro Glu Asp Gln 115 120 125
Ser Cys Thr Gln Gln Gly Ile Glu Trp Val Glu Pro Asp Asp Trp Thr 130
135 140 Ser Thr Leu Met Ser Arg Gly Arg Asn Arg Gln Pro Leu Val Leu
Gly 145 150 155 160 Asp Asn Val Phe Ala Asp Leu Val Gly Asn Trp Leu
Asp Leu Pro Glu 165 170 175 Leu Glu Lys Gly Gly Glu Arg Gly Glu Thr
Gly Gly Ser Gly Glu Pro 180 185 190 Lys Gly Glu Lys Gly Gln Ser Arg
Glu Leu Gly Arg Lys Phe Ala Leu 195 200 205 Thr Ala Asn Ile Phe Arg
Lys Phe Leu Arg Ser Val Arg Pro Asp Arg 210 215 220 Asp Arg Leu Leu
Lys Glu Lys Pro Gly Trp Met Thr Pro Met Val Ser 225 230 235 240 Glu
Ser Arg Ala Gly Arg Ser Lys Lys Val Lys Lys Arg Ser Leu Ser 245 250
255 Lys Gly Ser Gly Arg Phe Pro Phe Ser Ser Thr Gly Glu Pro Arg His
260 265 270 Ile Glu Thr Pro Ala Thr Ser Ser Pro Lys Ala Leu Glu Pro
Ser Cys 275 280 285 Arg Gly Phe Asp Ile Asn Thr Ala Val Trp Val 290
295 49 949 DNA Eukaryote misc_feature (1)...(949) r = A or G y = T
or C m = A or C k = G or T s = G or C w = A or T b = G, C, or T;
not A d = A, G, or T; not C h = A, C, or T; not G v = A, G, or C;
not T n = A, T, G, or C 49 tttktttkta attttttttt tttnatttgg
gttgattcct tgtnttttan ttgccaaatn 60 ttaccgatca ntgancaaag
caagcacagc caaaatcgga cctcacctta attccgtctt 120 cacacaaaaa
taaaaaaacg gcaaactcac ccccattttt aattttgttt ttaattttac 180
ttacttattt tatttattta ttttttggca aaaaaatctc aggaatggcc ctgggccacc
240 tactatatta atcattttga taacatgaaa aatgatgggc tcctcctaat
gaaaaascaa 300 ggaaaggaaa aggccagggg aatgagctca aaattgatgc
ccacktgggg agcatctggt 360 gaataatcgc tcacktcttt cttccacagt
accttgtttt gatcatttcc acagcacatt 420 tctcctccar aaacscgaaa
aacacaascg tktgggttct gcatttttaa ggataarara 480 raraaagagg
ttgggtatag taggacaggt tgtcagaaga gatgctgcta tggtcacgag 540
gggccggttt cacctgctat tgttgtcgcc tccttcagtt ccactgcctt tatgtcccct
600 cctctctctt gttttagctg ttacacatac agtaatacct gaatatccaa
cggtatagtt 660 cacaaggggg taatcaatgt taaatctaaa atagaattta
aaaaaaaaag attttgacat 720 aaaagagcct tgattttaaa aaaaaaagag
agagatgtaa tttaaaaagt ttattataaa 780 ttaaattcag caaaaatttg
ctacaaagta tagagaagta taaaataaaa gttatyhgtt 840 tcaaamtavc
dtrtcgamct cvtcvabccc grggaakccm ctaskkcbar hscggccccc 900
accscssysk akmtycatkc ttttgawwcc ctttagtgag ggttaanaa 949 50 785
DNA Eukaryote misc_feature (1)...(785) n = A,T,C or G 50 cagcctctca
ctctctngct ctctttctgt ctcttcctcg ctccctctct ttctctcctc 60
cctctgcctt cccagtgcat aaagtctctg tcgctcccgg aacttgttgg caatgcctat
120 ttttcagctt tcccccgcgt tctctaaact aactatttaa aggtctgcgg
tcgcaaatgg 180 tttgactaaa cgtaggatgg gacttaagtt gaacggcaga
tatatttcac tgatcctcgc 240 ggtgcaaata gcttacctgg tgcaggccgt
gagagcagca ggcaagtgcg atgcagtctt 300 taagggcttt tcagactgtt
tgctcaagct gggtgacagc atggccaact acccgcaggg 360 cctggacgac
aagacgaaca tcaagaccgt gtgcacatac tgggaggatt tccacagctg 420
cacggtcaca gctcttacgg attgccagga aggggcgaaa gatatgtggg ataaactgag
480 aaaagaatcg aaaaacctca atatccaagg cagcttattc gaactctgcg
gcagcggcaa 540 cggggcggcg gggtccctgc tcccggcgct ttccgtgctc
ctggtgtctc tctcggcagc 600 tttagcgacc tggctttcct tctgagcacg
gggccgggtc ccccctccgc tcacccaccc 660 acactcactc catgctcccg
gaaaatcgag aggaaagagc cattcgttct ctaaggacgt 720 tgttgattct
ctgttgatat tgaaaacact catatgggga ttgttgggna aatcctgttt 780 ctctc
785 51 782 DNA Eukaryote misc_feature (1)...(782) y = C or T m = A
or C k = G or T w = A or T n = A,T,C or G 51 aaaccnagaa cccccctttg
nagaaccntt gtttcctttc aagcccaagg aaggcggggc 60 ccaacctttg
gtgttntttg aacaggcctt gaacaggagg ntwaggagaa atttccggtt 120
gtggaacccc aacaggaacc ccttggcacc cctggcccca aggttgtgma actttggttt
180 gcttaatttg gaccgttttt gccttgagga ttcatgactt ttttttgkgc
ccttgtgagc 240 caagatgttg ggttttccca tcaacawtaa taaccccttg
ctttttgggg tgattcccct 300 ggggagtttc ctgatgaatt cccccacagc
tcctggggtt ttcatcttgt tcttactgtt 360 gtctggatta ggagggcgga
gagggtggac tccctgagac aagataagca ggtggagaca 420 tagaagaggg
agggacattt aacatagtaa cattttcaga ggtgacagag atgatacacg 480
ggcagctgga mttttgtgaa ggacagagga gctggcagac ccacagggcc atacctttga
540 gggacaggtg aatggctggt taccagagac aggactggta gacagtcaag
tacctcacta 600 cgatgtgcca agagatytgg gatcctggga aatgtgtgga
gaagaggatt tgacactccc 660 cacccccaag gcccttcccc tttgctgaca
gcattgctgt ggtcgtggcc tgttgccttg 720 tcctctgtcc ctgggtgggg
cacaccctcc tgtgctgtgc ttgccttgtg catcaataaa 780 cc 782 52 1613 DNA
Eukaryote misc_feature (1)...(1613) r = G or A n = A,T,C or G 52
gcantttgga gttattgctt aaaaccaggn taaggcactt tgtcccacag gacccaggaa
60 tcntaaangg gttgaaattg ggncggggaa ccccaggata taatgcnact
tttgttaggg 120 ggagagttca gctctaactg gtagtagtgt gaaagtaagc
accttgactt caattttgga 180 aagcacttgg taaatggaga gaactttgga
gtttccctat catctatatc agtctttgaa 240 cacaccctca agtcccagcc
tcaaggctca ataaaggacc acatagcagg tctgaggctc 300 actgctctca
gcccttaaca cagggcagtg gagagcaggg tgatcttccc tctctggagc 360
ttctccttgg ccttcttctc cacttgggct tctgctcagc agcagatata ttctgggttc
420 cataaggaat ccagctgtcc cagtggcttg accctgtcaa ggcaagatat
caactctgag 480 gatgacccag tcatggagga agagagtgtg acaagatccg
cagtttgaag caaaactgtg 540 tttggtcttt tcaagaaaca aatgggcaca
ttgagttctg ttcagtgtca gaggatatct 600 ttccctttgc tcccagattt
ccagaaatgg ataatgtttt catttctgtg ggaagggtca 660 agaaacataa
aattgctcaa caatgcttgc ttcccttgag ggttgttgag caaaggccga 720
tatgcctccc tgcattctct tctacctcaa gattttggaa ttcaattctg gaacagaaat
780 ttatttacac aagaacactt gttgtcagcc ttggttactg tgggagttac
ataagggtga 840 cagtctgtat cttctaartt aaacaggaac tgggctttgg
cggcctattg acccagttta 900 tatctaaata taactgtggc tccaaatgat
tggccaataa cattcccttt accttcaaag 960 ttttctccat cagtcatttc
tgtggcagca cagttccaat gtcatatgcc cctgcaaatt 1020 gtgaaagtaa
ttagtgacaa aataaccctc ccccctttca gtggccaaac tgtcagctgt 1080
agcagcgctg cgaaagcgag tactacacta tgtacggaaa gcctgttcct tatcacggac
1140 tagactcaag aaatgccatc tccgaacggt ggcattcaag gtggtagtcg
tttgaatgga 1200 acagtcatct atgtggacat tgttaaagtg ttttaaagag
tattttgaaa attaagttta 1260 cattttacaa ctgctttatt tttattgaaa
caattgtata taaatattac cctctttcac 1320 tgttaattaa agtaaaccta
gaccttgtag acaagtgggt caactgatat gtatagaagc 1380 tgtgatgtag
acaatacctt tctcttgtgt aaatggtcat aaatatagct gttcctgtgt 1440
ttttataagt tgagggtatt ttgttgtttt ataacaacaa aatttattgc atttgaaatg
1500 gtttttatgt aatagaatca tgcaaacagt gaaggattat aacatggtat
atgtaaatgt 1560 ataaacttta gaaagaaata aatacaacaa atttcaaaaa
aaaaaaaaaa aaa 1613 53 1669 DNA Eukaryote 53 ggcacgagga cagattctga
gatggaaact taaattacat cccagaggca gggaaactat 60 gaagtcaccg
ttcctagacc accccttact gaggttccac ggtcacactg acggcaggac 120
ccacaagggc agggtattgg tctgccctcc tttctcctgt ctgtctgact tacctaactt
180 tggtctcggc tgctgacact tggaaaggac caaattactt gatagtattt
ccccctgttt 240 gtgtaatagc ctgaaacctt ggagaggttc cagaatactt
ctgtatatag ggcacaggtg 300 aagacattgt ccaaagctta tttatttatc
tatttattta ccctggctga gtaaccacac 360 cagtaggggg aaaactaaaa
tgtgttgagt gtaaacaaag tcaccagcct ggctagaaat 420 tctccctgga
aaacatccat tttgatacaa tgtaaacgtt agtgttcacc cttagataca 480
tgttgaaaga gagctttggt acgcggaagt ggcatctttg gtcacacacc atgccaaagt
540 gaagaggtgg ccagtggagg tcttccggtc ctgtcgggat catttgtgaa
tacattcttt 600 gcccctctta agtacttgtt tactaaacat gtgcagtggt
aggtattagt gttagatcac 660 agtgggcact tccctgggga tctggggaag
accagagctt gcaactctgc ctgttttgat 720 ccctatttct cacagtgctg
tattaaaaaa aataggattt aagacagata accaccttta 780 cattgtgagt
gtgtttgcct tgtctaacga cagataattc cttaacattt ctcttcacct 840
tagtacttta ggctaattat acacgtctgt ctatgccatg agtaagtgga ctgtagtcgg
900 accaaaagaa aacaaatgag ccgttggacc atttgtgcag tcagtttctg
gtccttagat 960 gtatcctaag cagtaagtgt ctgattgtac cctggtggta
tgatcagttg tctcgtagct 1020 gtctcagctc cacagtttac aatgcaaatc
tgtctcaaga tcttcacgtc actgctgctg 1080 agagcaggga gaattctctg
cagctgtttc aaagttgtgg cccggccttg aatcctctgt 1140 taattactgt
gtgagccaga gggagctgcc cagcaagggt gggcccccag ccggcagggg 1200
aactttctag actccccgct cattcaattg atctaggcat tcgggcctgc tacttgacca
1260 ttctcgccct gtgaaatgtc ccacactttg aagcaaatac aattcacagc
acagtacaca 1320 caaaaaccct ggcataagac aggggaggtt cttcttattt
tgtgagccgg ttgccctgga 1380 aacggataac aaagggcagc cttccacttc
tggcataatg gtggagcctc ttttctcagg 1440 cttgacacct gtctgaataa
gagtgattag agccgcataa tatccctctc ttggctattg 1500 aatatgtggt
tcacatacca aaccctgtag aagttagaag acggtcgtgt tcgtatgttg 1560
tttgcttcca ctacattttt gaggttttgt aaaactgtta ttttttttca cgatgtgaaa
1620 ctgaaggtca ataaattatt agagattttc aaaaaaaaaa aaaaaaaaa 1669 54
1586 DNA Eukaryote misc_feature (1)...(1586) n = A,T,C or G 54
cttaaaaccc ctagatttcc tgttacatac taacacaggt cttccctttc actccaaccc
60 caggtttcag gcctcagagc catgctgggg ttggagaaaa ctgcattcct
atgagggtaa 120 aaagtagctg ccctctctga ccctttcttg ctaggcttca
tgcgggatgg gagagggtat 180 ccccaggatg gggacagagg aagcctggct
agggccttct agcccaataa gccaaacagg 240 aactataagc agatcaaaat
cctacactag cttattaggg ccctgttagt tgaaaacctt 300 gttgctgtcc
caagttcttc agttacaacc gagtacactt actcttccaa ctgtcctaag 360
ggtcactacc cagccagctt tggatcttca gcacttttaa aagctgaaac tccctcttgc
420 ccttcttgtc tattcctcac tgccagttgg ggcctaggct cagtcctggg
caaatgccca 480 tgatcctgct gctgtgggaa gtttgatagg gcatttggct
caaatttcaa aaggcctcgc 540 tcctgacctg atttctcgaa gctccagtag
ttctagaccc ctccaatctc tcatctgact 600 ggttgcaagg cttatttttc
ttttgtactt tcctatagag catttctgta gcatttgagt 660 gtggcgatat
ttttgttgtg tgtagatttc taagaaccaa cactactcag tctcctgcta 720
gtctgactcc tgaagcatca gacctcgtca tacggtattg actgtgtatg tgcctttcac
780 cttgagcatg cttcaggatt ttttttctta aaccacagaa cttgaataca
caagggaacc 840 agaattcaca aagtcctatg caaccctaga caggaggagg
ttagagagtc tgtcttgatt 900 ggtgatttca gagacccnag agaaatttgt
accagtttgt attaatgtca gtactaccag 960 cactttgcca aaactaagga
tgtcagaggg acctgtttct agagtgagtc ccaattacat 1020 caaagggcaa
cttacagctt tctccagtaa gtctgagtgg ttctcttgag ctggtgtcac 1080
tttctaacct ttgccagtct agcccagcag ggccctgtgt gtgtgagtgc agtttggtgc
1140 tgttttggag tatgcctgct ccccagcctg gaaccctctc agcaacttgc
tgggacctat 1200 aatgtcttag gtgcaacaag gaccctacca gagctcctgg
gtggctttca agatccacgt 1260 agctttgtgt gaggggactg aatgcagaca
aaccacagcc tgcttcaaat accttctttc 1320 cctaccacct agttccaaat
ggaaccaaca agttgagtgc atctctgttg ggtgttttgt 1380 gttgagactg
gctgaagtga aaactctttg actgaccatg ttgtgatgtg tcgacagact 1440
caaggacaca accacctcga gctggtcatg tggcatgcct gtgtatgtgt gtaacaggat
1500 tctgaatgtt aggttgtaat gctattcctg tatgggagaa aaaaataata
taaacaaata 1560 aaaatctatt taaagcacaa aaaaaa 1586 55 770 DNA
Eukaryote misc_feature (1)...(770) n = A,T,C or G 55 ccatggggac
tggtttgtca ccnattgccc atggnttggt tggtaggtgt tttttggtgg 60
acatttttgt ttcncgtttt gaactccaga ttattgggtt tttgttttaa tttatttttg
120 tcagaggaaa aataatttaa catccatctc acaggcttgc ttgactgttc
agttccaagg 180 tcctgctcac tttttcttgt cttgcctctg ctctggcttt
cttcatgata gtgctggacg 240 tggagctgag agtctcgttt actctaggca
aaccctctac ctgaagccag agcccagcac 300 tccgtaccac cacagacttc
tgaagctggc aaagttttag aagctgggag ttttctgatt 360 ctctcattat
taagtttctc ctcagtcttt agatagaggt aaatgtgggc ttgtaagaaa 420
agaaacgaaa gcacgtaatg tacacctatt ctgaattatg caaattagct cttactcagg
480 gtcaactaaa ttacttcaac tcgcccttta gtttactctt aatttgcaaa
aagagaaaaa 540 agaaggaaaa ctaaatagga ctatgatttg gggagccaaa
ttgataatct gatgtaaaag 600 ttgctgtgtt aaacataaat tattaagtgt
agactttttt cctaggatat tgtattcatt 660 ttgtgatatc gcctagaatg
atgtattaga taaaaatcaa ttttgtaagt atgtaaatat 720 gtcataaata
aatactttga cttatttctc aaaaaaaaaa aaaaaaaaaa 770 56 983 DNA
Eukaryote 56 gattttatat tcaatgttgt ttatttaatc cattgcagtt ggtgaatgcc
ttttcctcct 60 agacaccctg tattatacca tttggggatt aagtcaaagt
taagtatatt tttttcttac 120 ttgagctcta tatatgcaat tcagatatct
tcctgatgac agttttatat gtaaatgtaa 180 tttaactttc tttccgtgtt
gacgaagttc tgtaggtgtt agggttagaa gtctcagcac 240 tcacttctct
cactggatgt gcagtgtgcc tgccatggcg cacggcttct cagtaatgat 300
gccatctctg ctacttttac agaaggagaa gtttactttt gaggtgggta tgtgttgata
360 tctaaacact gtgtgttgct tgcttagata ggcaagacac actgctgtgc
gtggctcctg 420 tggtgcacct agcccagggg aacgtagcct cagtacttcc
gctggcttct tcatgcctaa 480 gaagcagggg cctttcttgt ttgctgggct
ctggctttaa aagttgtcct ttgggtctgg 540 agatgtagct ctgtgacaga
acaccagcta atgtcaggtc ctgcggtcag tctctggtac 600 acacaagcgc
acactcacat gatgggggga tgaaaggctg tccttgtgta acagtattcg 660
atggggcgtt gcctggatga cgatgtttat gtactctgaa ggcagatcct gaaggcaccc
720 tgttcttccc ttccttgtgt aactgagtct gcactagctt agccactgtt
ttagaggcca 780 tcctagtggg cgaacaggag gcatcgcact gggtgatggt
ttgccttcag tcctcaagta 840 acagcggccg acttatgccg atggcttgtt
tgaaatcaaa tattaccaag ttggcctagt 900 ctgccttctg tgaagaaggg
gagaaaggaa gggtggaaag gtggatggaa agcctttggg 960 gaactagtct
gatctctcaa ggg 983 57 1763 DNA Eukaryote misc_feature (1)...(1763)
y = C or T n = A,T,C or G 57 cttcagttcc tttgaggggn ctttccttcg
aaggggatac gcctaccttt cacgagttgc 60 gcagtttgtc tgcaagactc
tatgagaagc agataagcga taagtttgct caacatcttc 120 tcgggcataa
gtcggacacc atggcatcac agtatcgtga tgacagaggc agggagtggg 180
acaaaattga aatcaaataa tgattttatt ttgactgata gtgacctgtt cgttgcaaca
240 aattgataag caatgctttt ttataatgcc aacttagtat aaaaaagctg
aacgagaaac 300 gtaaaatgat ataaatatca atatattaaa ttagattttg
cataaaaaac agactacata 360 atactgtaaa acacaacata tgcagtcact
atgaatcaac tacttagatg gtattagtga 420 cctgtaacag agcattagcg
caaggtgatt tttgtcttct tgcgctaatt ttttgtcatc 480 aaacctgtcg
cactccagag aagcacaaag cctcgcaatc cagtgcaaag cttgcatgcc 540
tgcaggtcga ctcatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc
600 atcaggcggc catcgccctg atagacggtt tttcgccctt tgacgttgga
gtccacgttc 660 tttaatagtg gactcttgtt ccaaactgga acaacactca
accctatctc ggtctattct 720 tttgatttat aagggatttt gccgatttcg
gcctattggt taaaaaatga gctgatttaa 780 caaaaattta acgcgaattt
taacaaaata ttaacgctta caatttgcca ttcgccattc 840 aggctgcgca
actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg 900
gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca
960 cgacgttgta aaacgacggc cagtgaattg taatacgact cactataggg
cgaattgggt 1020 accgggcccc ccctcgaggt cgacggtatc gataagcttg
atatcgaatt cggcacgagc 1080 cgcagccgat atgcagtccc cggcggtgct
cgtcacctcc aggcaagttc agaatgcgca 1140 cacgggyctc gacctgactg
taccacagca ccaggaggtg cggggtaaga tgatgtcagg 1200 ccatgtggag
taccagatcc tggtggtgac ccggttggct gtgttcaagt cagccaagca 1260
ccggcccgag gatgtcgtcc agttcttggt ctccaaaaaa tacagcgaga tcgaggagtt
1320 ttaccagaaa ctgtacagtc gttacccaga agccagcctg cccccactgc
ctaggaaggt 1380 cctgtttgtc ggggagtctg acatccggga aaggagagcc
atgtttgatg agattctacg 1440 ctgtgtctcc aaggatgccc agttggcggg
cagcccagag ctgctagaat tcttaggcac 1500 caggtccccg ggggctacag
gctttgccac ccgagatccc tctgtcttgg gatgacgaca 1560 gccagccggc
caggggacag tgatgaggct tttgacttct ttgagcaaca gggatgaagt 1620
gcaagccacc cacattgggc ctgagcaaca angaaatgtt gagaaggtcc ntggaaggaa
1680 ngaggaggga agggaggaag gangataact tgggatcccc cttggggcaa
tcaatgcggc 1740 ctcccaaagg aaagncccta aag 1763 58 4634 DNA Rattus
norvegicus misc_feature (1)...(4634) r = G or A y = C or T m = A or
C k = G or T s = G or C w = A or T n = A,T,C or G 58 ctgccagccg
aggctcctgc cgctgtgacc cgcgctccgc ccgccgccgg gccgggaccc 60
tgatagctaa tgtcagaaga aagtgactct gtgagaacca gcccctctgt ggcctcactc
120 tccgaaaatg agctgccacc gcctcccccg gaacctcccr gctacgtgtg
ctcgctgaca 180 gaagacttgg tcaccaaggc cagggaagag cttcaggaga
agcccgagtg gagactccgg 240 gatgtgcagg cccttcgaga catggtacgg
aaggagtacc catacctgag tacatcgctg 300 gatgatgcct tcctgttgcg
ctttctgagg gcccgaaagt ttgattatga ccgggccctg 360 cagctgctgg
tcaactacca tggctgcagg cggagctggc cagaggtctt cagcaacctg 420
aggccatcag ccctgaaaga cgttcttaac tctggattcc tcacagtgct gccccacaca
480 gaccccaggg gctgccatgt cctctgcatc cgaccagaca gatggatacc
gagcaactac 540 ccgatcaccg agaacatccg cgccatctac ttgacgttag
aaaaactcat tcagtccgag 600 gagacccagg tgaacggggt tgtaatcctc
gccgactaca agggagtgag cttatcaaag 660 gcgtctcact ttggcccctt
tatcgccaga aaggtgattg gcatccttca ggatggcttc 720 cccattcgga
taaaagcagt tcacatagta aacgaacctc ggatatttaa gggcattttc 780
gccatcataa aaccatttct gaaggagaaa attgcaaaca ggttcttcct ccatgggtct
840 gacctgagct ctctgcacac gagccttcca aggaatatcc tccccaaaga
gtatgggggc 900 accgctgggg agctggacac tgccagctgg aacgcggtgc
tgctggcctc ggaggatgat 960 tttgtgaaag agttctgcca gcctgagtct
ggctgcgatg gtctcttggg ccagcccctg 1020 ctgcctgagg ggctgatctc
agacgcgcag tgtgacgact ccatgcgagc catgaagtcc 1080 cagctctact
cctgctatta gccctcttcc gggagaatca ccatgtgtaa ttccttcctt 1140
cttcgaatgc acaggctgaa gatgccagga cctcggtctt gctccatcac agtgcagcac
1200 ggagctgcct gcagagattt aaggagagcc catcacaggc agacctctga
ccagctaggt 1260 tattccaaga agacatggaa attgccctgg tgattcccag
atgtctgtac tctaagtctg 1320 caactgttac tctggaagct gcatctgttt
cttatgcatc ttggaaagaa ctagggtcaa 1380 agtcactctg aagtgaccag
gagtagacaa cttgattgat catgagtctg aaacaattgc 1440 caatcctgaa
aggtggccat gcgtgagact ttgagtctct ttcccataaa ctgtaggtgt 1500
tgactactgc tgcttatctg caaaggtcag ggttcaggcc ccagttggca ttgctgggtc
1560 tgggaagcac tgctaactga gtggtagaaa cgccaggccc aggcagcact
taaaggttaa 1620 aggtcaaatt tggaagctaa ggctataaat catcctgggt
tccaggctta aatcttgcaa 1680 tggacactct ccccaaacca taaagcctta
gctctggttc tccatggaat catgcaggtc 1740 aacataaaat actggattct
tggactgcgt ggctaaaagc acttagacta rgagtccagt 1800 gtgtgactgg
atggataggg gcctcagctt gtcaactcta agttagmgmt ccatggaatg 1860
aaggccttgr gggctgctca agttctgtta ggtttctgct tggaaagatg accacctgga
1920 ggtggccggg cctttttggt ttggcttggt tttgtgttat agacacaagc
cttatggaaa 1980 ggaaccgtct ggcctttaaa gaaattacta tgttcctggg
agttggtggt aaccagctgc 2040 ttttgcagat gatgggtgaa ctggaaaggg
atggcttttg tgaggctgac caagtcttgt 2100 acgcggatgt tgtacagatt
cctcccacac cggagacatt cgtactatat tagaaacagc 2160 cacggacttg
tgctctttca gtttgtgtcc ctggaaacat acggggggca ggctgttgct 2220
ggttcacctg ggggccctgc cctcccagac acgggagtgc ttgtctagcg tgggagggcc
2280 agttggccag attgttagct ctgcgttggg gtgtcgtaga caactgacag
gattttagcc 2340 ttaacccaag cactgagtga ggtgattttt cccttggctt
ttggcgtgtc tttggtattc 2400 accatgtatt gtggtgtcag gtagtgtcag
gtactgttgg ctgtgtgtct cctagactaa 2460 gcgggcgttg satacagctt
acatacagtg cttggagacc aaaggtcagt tggttgtaat 2520 aagctggtcc
acccttaaca gacttcccaa acatyacaga agctyttatg gmccttacct 2580
aataatgcca attctggagg acactctttt accatagawk csaatccttg atctcctggc
2640 tcctggttga gcttccgcac tgatacaccc tcttgrctgc ccatcagggc
catttgctgc 2700 tgagttctgc attgcttaak ctsckgsygy tttctgccta
aagggatggc cacccagaca 2760 cctaaaaaga cccgggatgg ctctctagcc
ttggtggaga gtcttattag aagttttctt 2820 tgggggattg gggatttggc
tcagtggtag agcgcttgcc tggcaagcac aaggccctgg 2880 gttcggtccc
cagctcttaa aaaaaaaaaa agttttcttt ggtagttggg gaaaaggcag 2940
aaggaaaaaa acaaagggaa agatgaatct ctcagtccta cctggttccc taaatttaaa
3000 tcgtgtcatg tgactagtta agtctctttg acttaacaaa gggacaccag
gttcttgggg 3060 agaaatctca gagcaaaatg ttgcctgttg staaccttct
ggtaaccara ggarccttga 3120 taarcttarg agykgactgt atgtccatgc
tcttgtgact ctagagactc tggcacctca 3180 ggttnaagca ggctgtgagc
cagatgtcct ggtgccaagc aaccccactg ttgagcagca 3240 ggggcaccat
aggcctcagc taggggagcg cactggtaga gccagcaagt gagcaggaat 3300
ctgactttag ggtaaaaatc tagacagttc tgacagctgg aagtcaactt ttcctccatt
3360 caaagtcatg tggcattggg aaggggctag ggaaatagaa gtgggttcca
gctttatctt 3420 cctacacagt ctcgagtata gcattaacac cgagtgctgg
acagaggttg tctgctgaac 3480 actcaatcct gctcctgact gactctggaa
ataaggacat tccactctgc ttggcgcgga 3540 gatgccctag tgtgcggccg
cgggggcttc tctttctcaa gtcctctaca gnacttccag 3600 gcagttcatc
ttcctaggaa aaggtatgga ggttctgcct tcatggtaga aacacaggat 3660
aaaatctaca gtaaacaacc ggtaagtgct ggcttcttac gccttggctt tctccaggca
3720 caggtgggtt cgactactcc catttcatct ttgtaagcac ctcaggttat
agggcagttt 3780 cttcagagtt ggggggactg gagccattcc ccctgtaatg
cctgaggtgg ccttaccacc 3840 tagcagccag tttggccagc aacagccaca
ctgctgttat ggtatcataa tacctcatcc 3900 tcgggtttcc ttcagaaagg
raaawgctaa ctcagttgat gtaagtgttg ctgtgctggg 3960 atcctgtcat
gtgggaggga acaccaaata cacaggctct caggagacat cttgctaagg 4020
cttctcttta ctgcagtctg ctcacgttgt aaatctgccc tctgttctcc tgactcaraa
4080 agactcagcc mcaaatcaag aagcgccatc aaacgttcct tctcakkggg
aacgtgctcc 4140 acaggaaggt ccagwgggat ttgcarctag agtcacgttt
tactggkttg tgamcaaatt 4200 tactggtttt carttacctg gggkcctatg
kgkkttttma accttttccc atmaggcagt 4260 tagtagtagc cactttgggt
tcctgtggac gtgcctcagc ttctcggcat aggaacccaa 4320 caggtagaat
acttgaaact tctcagtggc caagacctcg ataccctctc tgatgggtgg 4380
gaactgggct attttcctga ccaatctagg ccaccatttt agtccctggt cacattcctt
4440 actccaaact gaaattcagt ttggctttga gtatgtgcac acgtggtggg
ttcacctact 4500 tcagtgttga ccaaaagttt atttttctag tgcatttttc
taaatggtaa aaatatgtaa 4560 ttttagtatg catgactggg tctccaaaat
aaaaactgag tgtattgtga aaaaaaaaaa 4620 aaaaaaaaaa aaaa 4634 59 1030
DNA Rattus norvegicus CDS (1)...(1030) 59 atg tca gaa gaa agt gac
tct gtg aga acc agc ccc tct gtg gcc tca 48 Met Ser Glu Glu Ser Asp
Ser Val Arg Thr Ser Pro Ser Val Ala Ser 1 5 10 15 ctc tcc gaa aat
gag ctg cca ccg cct ccc ccg gaa cct ccc ggc tac 96 Leu Ser Glu Asn
Glu Leu Pro Pro Pro Pro Pro Glu Pro Pro Gly Tyr 20 25 30 gtg tgc
tcg ctg aca gaa gac ttg gtc acc aag gcc agg gaa gag ctt 144 Val Cys
Ser Leu Thr Glu Asp Leu Val Thr Lys Ala Arg Glu Glu Leu 35 40 45
cag gag aag ccc gag tgg aga ctc cgg gat gtg cag gcc ctt cga gac 192
Gln Glu Lys Pro Glu Trp Arg Leu Arg Asp Val Gln Ala Leu Arg Asp 50
55 60 atg gta cgg aag gag tac cca tac ctg agt aca tcg ctg gat gat
gcc 240 Met Val Arg Lys Glu Tyr Pro Tyr Leu Ser Thr Ser Leu Asp Asp
Ala 65 70 75 80 ttc ctg ttg cgc ttt ctg agg gcc cga aag ttt gat tat
gac cgg gcc 288 Phe Leu Leu Arg Phe Leu Arg Ala Arg Lys Phe Asp Tyr
Asp Arg Ala 85 90 95 ctg cag ctg ctg gtc aac tac cat ggc tgc agg
cgg agc tgg cca gag 336 Leu Gln Leu Leu Val Asn Tyr His Gly Cys Arg
Arg Ser Trp Pro Glu 100 105 110 gtc ttc agc aac ctg agg cca tca gcc
ctg aaa gac gtt ctt aac tct 384 Val Phe Ser Asn Leu Arg Pro Ser Ala
Leu Lys Asp Val Leu Asn Ser 115 120 125 gga ttc ctc aca gtg ctg ccc
cac aca gac ccc agg ggc tgc cat gtc 432 Gly Phe Leu Thr Val Leu Pro
His Thr Asp Pro Arg Gly Cys His Val 130 135 140 ctc tgc atc cga cca
gac aga tgg ata ccg agc aac tac ccg atc acc 480 Leu Cys Ile Arg Pro
Asp Arg Trp Ile Pro Ser Asn Tyr Pro Ile Thr 145 150 155 160 gag aac
atc cgc gcc atc tac ttg acg tta gaa aaa ctc att cag tcc 528 Glu Asn
Ile Arg Ala Ile Tyr Leu Thr Leu Glu Lys Leu Ile Gln Ser 165 170 175
gag gag acc cag gtg aac ggg gtt gta atc ctc gcc gac tac aag gga 576
Glu Glu Thr Gln Val Asn Gly Val Val Ile Leu Ala Asp Tyr Lys Gly 180
185 190 gtg agc tta tca aag gcg tct cac ttt ggc ccc ttt atc gcc aga
aag 624 Val Ser Leu Ser Lys Ala Ser His Phe Gly Pro Phe Ile Ala Arg
Lys 195 200 205 gtg att ggc atc ctt cag gat ggc ttc ccc att cgg ata
aaa gca gtt 672 Val Ile Gly Ile Leu Gln Asp Gly Phe Pro Ile Arg Ile
Lys Ala Val 210 215 220 cac ata gta aac gaa cct cgg ata ttt aag ggc
att ttc gcc atc ata 720 His Ile Val Asn Glu Pro Arg Ile Phe Lys Gly
Ile Phe Ala Ile Ile 225 230 235 240 aaa cca ttt ctg aag gag aaa att
gca aac agg ttc ttc ctc cat ggg 768 Lys Pro Phe Leu Lys Glu Lys Ile
Ala Asn Arg Phe Phe Leu His Gly 245 250 255 tct gac ctg agc tct ctg
cac acg agc ctt cca agg aat atc ctc ccc 816 Ser Asp Leu Ser Ser Leu
His Thr Ser Leu Pro Arg Asn Ile Leu Pro 260 265 270 aaa gag tat ggg
ggc acc gct ggg gag ctg gac act gcc agc tgg aac 864 Lys Glu Tyr Gly
Gly Thr Ala Gly Glu Leu Asp Thr Ala Ser Trp Asn 275 280 285 gcg gtg
ctg ctg gcc tcg gag gat gat ttt gtg aaa gag ttc tgc cag 912 Ala Val
Leu Leu Ala Ser Glu Asp Asp Phe Val Lys Glu Phe Cys Gln 290 295 300
cct gag tct ggc tgc gat ggt ctc ttg ggc cag ccc ctg ctg cct gag 960
Pro Glu Ser Gly Cys Asp Gly Leu Leu Gly Gln Pro Leu Leu Pro Glu 305
310 315 320 ggg ctg atc tca gac gcg cag tgt gac gac tcc atg cga gcc
atg aag 1008 Gly Leu Ile Ser Asp Ala Gln Cys Asp Asp Ser Met Arg
Ala Met Lys 325 330 335 tcc cag ctc tac tcc tgc tat t 1030 Ser Gln
Leu Tyr Ser Cys Tyr 340 60 1029 DNA Homo sapiens CDS (1)...(1029)
60 atg tcc gaa gaa agg gac tct ctg aga acc agc cct tct gtg gcc tca
48 Met Ser Glu Glu Arg Asp Ser Leu Arg Thr Ser Pro Ser Val Ala Ser
1 5 10 15 ctc tct gaa aat gag ctg cca cca cca cct gag cct ccg ggc
tat gtg 96 Leu Ser Glu Asn Glu Leu Pro Pro Pro Pro Glu Pro Pro Gly
Tyr Val 20 25 30 tgc tca ctg aca gaa gac ctg gtc acc aaa gcc cgg
gaa gag ctg cag 144 Cys Ser Leu Thr Glu Asp Leu Val Thr Lys Ala Arg
Glu Glu Leu Gln 35 40 45 gaa aag ccg gaa tgg aga ctt cga gat gtg
cag gcc ctt cgt gac atg 192 Glu Lys Pro Glu Trp Arg Leu Arg Asp Val
Gln Ala Leu Arg Asp Met 50 55 60 gtg cgg aag gag tac ccc aac ctg
agc aca tcc ctc gac gat gcc ttc 240 Val Arg Lys Glu Tyr Pro Asn Leu
Ser Thr Ser Leu Asp Asp Ala Phe 65 70 75 80 ctg ctg cgc ttc ctc cga
gcc cgc aag ttt gat tac gac cgg gcc ctg 288 Leu Leu Arg Phe Leu Arg
Ala Arg Lys Phe Asp Tyr Asp Arg Ala Leu 85 90 95 cag ctc ctc gtc
aac tac cac agc tgt aga aga agc tgg ccc gaa gtc 336 Gln Leu Leu Val
Asn Tyr His Ser Cys Arg Arg Ser Trp Pro Glu Val 100 105 110 ttc aat
aac ttg aag cca tca gcc tta aaa gat gtc ctt gct tcc ggg 384 Phe Asn
Asn Leu Lys Pro Ser Ala Leu Lys Asp Val Leu Ala Ser Gly 115 120 125
ttc ctc acc gtg ctg ccc cac act gac ccc agg ggc tgc cat gtc gtc 432
Phe Leu Thr Val Leu Pro His Thr Asp Pro Arg Gly Cys His Val Val 130
135 140 tgc atc cgc cca gac aga tgg ata cca agc aac tat cca att act
gaa 480 Cys Ile Arg Pro Asp Arg Trp Ile Pro Ser Asn Tyr Pro Ile Thr
Glu 145 150 155 160 aac atc cga gcc ata tac ttg acc tta gaa aaa ctc
att cag tct gaa 528 Asn Ile Arg Ala Ile Tyr Leu Thr Leu Glu Lys Leu
Ile Gln Ser Glu 165 170 175 gaa acc cag gtg aat gga att gta att ctt
gca gac tac aaa gga gtg 576 Glu Thr Gln Val Asn Gly Ile Val Ile Leu
Ala Asp Tyr Lys Gly Val 180 185 190 agt tta tca aaa gca tct cac ttt
ggc cct ttt ata gcc aaa aag gtg 624 Ser Leu Ser Lys Ala Ser His Phe
Gly Pro Phe Ile Ala Lys Lys Val 195 200 205 att ggc atc ctc cag gat
ggt ttc ccc att cgg ata aaa gca gtc cat 672 Ile Gly Ile Leu Gln Asp
Gly Phe Pro Ile Arg Ile Lys Ala Val His 210 215 220 gtg gtg aat gaa
cct cga ata ttt aaa ggc att ttt gcc atc ata aaa 720 Val Val Asn Glu
Pro Arg Ile Phe Lys Gly Ile Phe Ala Ile Ile Lys 225 230 235 240 cca
ttt cta aag gag aaa ata gca aac aga ttc ttc ctc cat ggg tct 768 Pro
Phe Leu Lys Glu Lys Ile Ala Asn Arg Phe Phe Leu His Gly Ser 245 250
255 gac ttg aac tct ctc cac aca aac ctt cca aga agc atc ctc ccc aag
816 Asp Leu Asn Ser Leu His Thr Asn Leu Pro Arg Ser Ile Leu Pro Lys
260 265 270 gag tat ggg ggc acg gct ggg gag ctg gac act gcc acc tgg
aac gca 864 Glu Tyr Gly Gly Thr Ala Gly Glu Leu Asp Thr Ala Thr Trp
Asn Ala 275 280 285 gta ctg ctg gct tca gaa gac gat ttt gtg aaa gag
ttc tgc caa cct 912 Val Leu Leu Ala Ser Glu Asp Asp Phe Val Lys Glu
Phe Cys Gln Pro 290 295 300 gtt cct gcc tgt gac agc atc ctg ggc cag
acg ctg ctg ccc gag ggc 960 Val Pro Ala Cys Asp Ser Ile Leu Gly Gln
Thr Leu Leu Pro Glu Gly 305 310 315 320 ctg acc tca gat gca cag tgt
gac gac tcc ttg cga gct gtg aag tca 1008 Leu Thr Ser Asp Ala Gln
Cys Asp Asp Ser Leu Arg Ala Val Lys Ser 325 330 335 cag ctg tac tcc
tgc tac tag 1029 Gln Leu Tyr Ser Cys Tyr * 340 61 341 PRT Rattus
norvegicus VARIANT (1)...(341) Xaa = Any Amino Acid 61 Met Ser Glu
Glu Ser Asp Ser Val Arg Thr Ser Pro Ser Val Ala Ser 1 5 10 15 Leu
Ser Glu Asn Glu Leu Pro Pro Pro Pro Pro Glu Pro Pro Xaa Tyr 20 25
30 Val Cys Ser Leu Thr Glu Asp Leu Val Thr Lys Ala Arg Glu Glu Leu
35 40 45 Gln Glu Lys Pro Glu Trp Arg Leu Arg Asp Val Gln Ala Leu
Arg Asp 50 55 60 Met Val Arg Lys Glu Tyr Pro Tyr Leu Ser Thr Ser
Leu Asp Asp Ala 65 70 75 80 Phe Leu Leu Arg Phe Leu Arg Ala Arg Lys
Phe Asp Tyr Asp Arg Ala 85 90 95 Leu Gln Leu Leu Val Asn Tyr His
Gly Cys Arg Arg Ser Trp Pro Glu 100 105 110 Val Phe Ser Asn Leu Arg
Pro Ser Ala Leu Lys Asp Val Leu Asn Ser 115 120 125 Gly Phe Leu Thr
Val Leu Pro His Thr Asp Pro Arg Gly Cys His Val 130 135 140 Leu Cys
Ile Arg Pro Asp Arg Trp Ile Pro Ser Asn
Tyr Pro Ile Thr 145 150 155 160 Glu Asn Ile Arg Ala Ile Tyr Leu Thr
Leu Glu Lys Leu Ile Gln Ser 165 170 175 Glu Glu Thr Gln Val Asn Gly
Val Val Ile Leu Ala Asp Tyr Lys Gly 180 185 190 Val Ser Leu Ser Lys
Ala Ser His Phe Gly Pro Phe Ile Ala Arg Lys 195 200 205 Val Ile Gly
Ile Leu Gln Asp Gly Phe Pro Ile Arg Ile Lys Ala Val 210 215 220 His
Ile Val Asn Glu Pro Arg Ile Phe Lys Gly Ile Phe Ala Ile Ile 225 230
235 240 Lys Pro Phe Leu Lys Glu Lys Ile Ala Asn Arg Phe Phe Leu His
Gly 245 250 255 Ser Asp Leu Ser Ser Leu His Thr Ser Leu Pro Arg Asn
Ile Leu Pro 260 265 270 Lys Glu Tyr Gly Gly Thr Ala Gly Glu Leu Asp
Thr Ala Ser Trp Asn 275 280 285 Ala Val Leu Leu Ala Ser Glu Asp Asp
Phe Val Lys Glu Phe Cys Gln 290 295 300 Pro Glu Ser Gly Cys Asp Gly
Leu Leu Gly Gln Pro Leu Leu Pro Glu 305 310 315 320 Gly Leu Ile Ser
Asp Ala Gln Cys Asp Asp Ser Met Arg Ala Met Lys 325 330 335 Ser Gln
Leu Tyr Ser 340 62 342 PRT Homo sapiens 62 Met Ser Glu Glu Arg Asp
Ser Leu Arg Thr Ser Pro Ser Val Ala Ser 1 5 10 15 Leu Ser Glu Asn
Glu Leu Pro Pro Pro Pro Glu Pro Pro Gly Tyr Val 20 25 30 Cys Ser
Leu Thr Glu Asp Leu Val Thr Lys Ala Arg Glu Glu Leu Gln 35 40 45
Glu Lys Pro Glu Trp Arg Leu Arg Asp Val Gln Ala Leu Arg Asp Met 50
55 60 Val Arg Lys Glu Tyr Pro Asn Leu Ser Thr Ser Leu Asp Asp Ala
Phe 65 70 75 80 Leu Leu Arg Phe Leu Arg Ala Arg Lys Phe Asp Tyr Asp
Arg Ala Leu 85 90 95 Gln Leu Leu Val Asn Tyr His Ser Cys Arg Arg
Ser Trp Pro Glu Val 100 105 110 Phe Asn Asn Leu Lys Pro Ser Ala Leu
Lys Asp Val Leu Ala Ser Gly 115 120 125 Phe Leu Thr Val Leu Pro His
Thr Asp Pro Arg Gly Cys His Val Val 130 135 140 Cys Ile Arg Pro Asp
Arg Trp Ile Pro Ser Asn Tyr Pro Ile Thr Glu 145 150 155 160 Asn Ile
Arg Ala Ile Tyr Leu Thr Leu Glu Lys Leu Ile Gln Ser Glu 165 170 175
Glu Thr Gln Val Asn Gly Ile Val Ile Leu Ala Asp Tyr Lys Gly Val 180
185 190 Ser Leu Ser Lys Ala Ser His Phe Gly Pro Phe Ile Ala Lys Lys
Val 195 200 205 Ile Gly Ile Leu Gln Asp Gly Phe Pro Ile Arg Ile Lys
Ala Val His 210 215 220 Val Val Asn Glu Pro Arg Ile Phe Lys Gly Ile
Phe Ala Ile Ile Lys 225 230 235 240 Pro Phe Leu Lys Glu Lys Ile Ala
Asn Arg Phe Phe Leu His Gly Ser 245 250 255 Asp Leu Asn Ser Leu His
Thr Asn Leu Pro Arg Ser Ile Leu Pro Lys 260 265 270 Glu Tyr Gly Gly
Thr Ala Gly Glu Leu Asp Thr Ala Thr Trp Asn Ala 275 280 285 Val Leu
Leu Ala Ser Glu Asp Asp Phe Val Lys Glu Phe Cys Gln Pro 290 295 300
Val Pro Ala Cys Asp Ser Ile Leu Gly Gln Thr Leu Leu Pro Glu Gly 305
310 315 320 Leu Thr Ser Asp Ala Gln Cys Asp Asp Ser Leu Arg Ala Val
Lys Ser 325 330 335 Gln Leu Tyr Ser Cys Tyr 340
* * * * *