U.S. patent application number 10/776604 was filed with the patent office on 2005-01-06 for isolated dna or gene responsible for parkinson's disease.
This patent application is currently assigned to Boehringer Ingelheim International GmbH. Invention is credited to Mizuno, Yoshikuni, Shimizu, Nobuyoshi.
Application Number | 20050003385 10/776604 |
Document ID | / |
Family ID | 12223704 |
Filed Date | 2005-01-06 |
United States Patent
Application |
20050003385 |
Kind Code |
A1 |
Shimizu, Nobuyoshi ; et
al. |
January 6, 2005 |
Isolated DNA or gene responsible for Parkinson's disease
Abstract
This invention provides an isolated DNA or gene that is
responsible for Parkinson's disease and is useful in diagnosing and
treating the disease etc. The isolated DNA or gene according to
this invention comprises a full-length base sequence according to
the sequence ID. No. 1 or 3, or a partial sequence thereof, or a
base sequence hybridizable thereto or hybridizable with a
complementary strand thereof, and being associated with Parkinson's
disease.
Inventors: |
Shimizu, Nobuyoshi;
(Chiba-ken, JP) ; Mizuno, Yoshikuni; (Tokyo,
JP) |
Correspondence
Address: |
STERNE, KESSLER, GOLDSTEIN & FOX PLLC
1100 NEW YORK AVENUE, N.W.
WASHINGTON
DC
20005
US
|
Assignee: |
Boehringer Ingelheim International
GmbH
Ingelheim am Rhein
DE
|
Family ID: |
12223704 |
Appl. No.: |
10/776604 |
Filed: |
February 12, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10776604 |
Feb 12, 2004 |
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09601844 |
Aug 9, 2000 |
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6716621 |
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09601844 |
Aug 9, 2000 |
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PCT/JP99/00545 |
Feb 9, 1999 |
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Current U.S.
Class: |
435/6.11 ;
435/6.12 |
Current CPC
Class: |
C07K 14/47 20130101;
A61K 38/00 20130101; A61P 25/16 20180101 |
Class at
Publication: |
435/006 |
International
Class: |
C12Q 001/68 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 9, 1998 |
JP |
10/27531 |
Claims
1-42 (Cancelled).
43. A method of detecting a neurological disorder in a patient, the
method comprising: (a) obtaining DNA from the patient; (b) creating
a mixture by contacting the DNA with a polynucleotide having a
nucleotide sequence that is either identical or complementary to a
nucleotide sequence which is adjacent to an exon of SEQ ID NO:1
under conditions which promote specific hybridization between the
polynucleotide and the DNA; and (c) detecting whether hybridization
between the polynucleotide and the DNA has occurred.
44. The method of claim 43, wherein the neurological disorder is
Parkinsonism.
45. The method of claim 44, wherein the neurological disorder is
autosomal recessive juvenile Parkinsonism.
46. The method of claim 43, wherein a polymerase chain reaction is
used on the mixture of (b) to amplify a portion of the DNA and
determining if the portion of the DNA is amplified.
47. The method of claim 46, wherein the neurological disorder is
Parkinsonism.
48. The method of claim 47, wherein the neurological disorder is
autosomal recessive juvenile Parkinsonism.
49. The method of claim 43, wherein the adjacent sequence is
adjacent to exon 1.
50. The method of claim 43, wherein the adjacent sequence is
adjacent to exon 2.
51. The method of claim 43, wherein the adjacent sequence is
adjacent to exon 3.
52. The method of claim 43, wherein the adjacent sequence is
adjacent to exon 4.
53. The method of claim 43, wherein the adjacent sequence is
adjacent to exon 5.
54. The method of claim 43, wherein the adjacent sequence is
adjacent to exon 6.
55. The method of claim 43, wherein the adjacent sequence is
adjacent to exon 7.
56. The method of claim 43, wherein the adjacent sequence is
adjacent to exon 8.
57. The method of claim 43, wherein the adjacent sequence is
adjacent to exon 9.
58. The method of claim 43, wherein the adjacent sequence is
adjacent to exon 10.
59. The method of claim 43, wherein the adjacent sequence is
adjacent to exon 11.
60. The method of claim 43, wherein the adjacent sequence is
adjacent to exon 12.
61. The method of claim 43, wherein the polynucleotide in (b) is
between 10 to 25 nucleotides in length.
62. A method of detecting a neurological disorder in a patient, the
method comprising: (a) obtaining DNA from the patient; (b) creating
a mixture under conditions which promote specific hybridization by
contacting the DNA with a polynucleotide having a nucleotide
sequence selected from the group consisting of: i. SEQID NO:31; ii.
SEQ ID NO:32; iii. SEQ ID NO:33; iv. SEQ ID NO:34; V. SEQ ID NO:35;
vi. SEQ ID NO:36; vii. SEQ ID NO:37; viii. SEQ ID NO:38; ix. SEQ ID
NO:39; x. SEQ ID NO:40; xi. SEQ ID NO:41; xii. SEQ ID NO:42; xiii.
SEQ ID NO:43; xiv. SEQ ID NO:44; xv. SEQ ID NO:45; xvi. SEQ ID
NO:46; xvii. SEQ ID NO:47; xviii. SEQ ID NO:48; xix. SEQ ID NO:49;
xx. SEQ ID NO:50; xxi. SEQ ID NO:51; xxii. SEQ ID NO:52; xxiii. SEQ
ID NO:53; xxiv. SEQ ID NO:54; xxv. SEQ ID NO:55; xxvi. SEQ ID
NO:56; xxvii. SEQ ID NO:57; xxviii. SEQ ID NO:58; xxix. SEQ ID
NO:59; xxx. nucleotides 351-371 of SEQ ID NO:1; xxxi. SEQ ID NO:70;
and xxxii. a nucleotide sequence complementary to any one of the
nucleotide sequences of (i), (ii), (iii), (iv), (v), (vi), (vii),
(viii), (ix), (x), (xi), (xii), (xiii), (xiv), (xv), (xvi), (xvii),
(xviii), (xix), (xx), (xxi), (xxii), (xxiii), (xxiv), (xxv),
(xxvi), (xxvii), (xxviii), (xxix), (xxx), and (xxxi); and (c)
detecting whether hybridization between the polynucleotide and the
DNA has occurred.
63. The method of claim 62, wherein the neurological disorder is
Parkinsonism.
64. The method of claim 63, wherein the neurological disorder is
autosomal recessive juvenile Parkinsonism.
65. The method of claim 62, wherein a polymerase chain reaction is
used on the mixture of (b) to amplify a portion of the DNA and
determining if the portion of the DNA is amplified.
66. The method of claim 65, wherein the neurological disorder is
Parkinsonism.
67. The method of claim 66, wherein the neurological disorder is
autosomal recessive juvenile Parkinsonism.
68. A method of detecting a neurological disorder in a patient, the
method comprising: (a) obtaining DNA from the patient; (b) creating
a mixture by contacting the DNA with a polynucleotide having a
nucleotide sequence that is either identical or complementary to a
nucleotide sequence which is adjacent to an exon of SEQ ID NO:3
under conditions which promote specific hybridization between the
polynucleotide and the DNA; and (c) detecting whether hybridization
between the polynucleotide and the DNA has occurred.
69. The method of claim 68, wherein a polymerase chain reaction is
used on the mixture of (b) to amplify a portion of the DNA and
determining if the portion of the DNA is amplified.
70. A method of determining a predisposition for a neurological
disorder in a human subject, the method comprising: (a) obtaining a
nucleic acid sample from the human subject; (b) assaying the
nucleic acid sample to determine the presence or absence of a
Parkin gene mutation associated with the neurological disorder,
wherein the Parkin gene mutation is a partial or complete deletion
of at least one of exons 3, 4, 5, 6 and 7 of SEQ ID NO:1, and
wherein the presence of the Parkin gene mutation indicates that the
human subject has a predisposition for the neurological
disorder.
71. The method of claim 70, wherein the neurological disorder is
Parkinsonism.
72. The method of claim 71, wherein the neurological disorder is
autosomal recessive juvenile Parkinsonism.
73. The method of claim 70, wherein the assaying in (b) comprises
contacting the nucleic acid with a polynucleotide hybridizable with
the nucleic acid and carrying out a polymerase chain reaction.
74. A method of determining a predisposition for a neurological
disorder in a human subject, the method comprising: (a) obtaining a
nucleic acid sample from the human subject; (b) assaying the
nucleic acid sample to determine the presence or absence of a
Parkin gene mutation associated with the neurological disorder,
wherein the Parkin gene mutation is a partial or complete deletion
of at least one of exons 3, 4, 6 and 7 of SEQ ID NO:3, and wherein
the presence of the Parkin gene mutation indicates that the human
subject has a predisposition for the neurological disorder.
75. The method of claim 74, wherein the neurological disorder is
Parkinsonism.
76. The method of claim 75, wherein the neurological disorder is
autosomal recessive juvenile Parkinsonism.
77. The method of claim 74, wherein the assaying in (b) comprises
contacting the nucleic acid with a polynucleotide hybridizable with
the nucleic acid and carrying out a polymerase chain reaction.
78. A method of determining a predisposition of a human subject for
Parkinson's disease, the method comprising: (a) obtaining a nucleic
acid sample from the human subject; and (b) determining whether a
point mutation is present at position 366 of SEQ ID No:1 or SEQ ID
NO:3, wherein the presence of a point mutation at position 366 of
SEQ ID NO:1 or SEQ ID NO:3 indicates a predisposition for
Parkinson's disease.
79. The method of claim 78, wherein the point mutation is an Arg to
Trp mutation.
Description
[0001] This application is a divisional of U.S. application Ser.
No. 09/601,844, filed Aug. 9, 2000, now U.S. Pat. No. 6,716,621,
which is a 35 U.S.C. .sctn. 371 national stage application of the
international application, PCT/JP99/00545, filed Feb. 9, 1999,
which claims priority benefit to Japanese application no. 10/27531,
filed Feb. 9, 1998, the full disclosure of which are herein
incorporated by reference.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates to a gene responsible for
onset of Parkinson's disease. Since it was found that Parkinson's
disease patients have a deletion in part of the gene, the gene of
this invention is significantly useful as a gene for diagnosing
Parkinson's disease, and a protein and a pharmaceutically active
agent etc., obtainable from the inventive gene has usability in
preventing and treating Parkinson's disease.
[0004] 2. Related Art
[0005] Generally, it is often considered that one or more gene is
responsible for various chronic progressive diseases. Isolating the
gene or genes responsible for these diseases not only enables one
to facilitate prenatal or postnatal diagnosis but also enables one
to perform gene therapy for the disease based on the remarkable
progress and development of gene therapy as seen today.
[0006] Parkinson's disease is a chronic disease. .alpha.-synuclein
reported in 1997 has so far been the only gene that has been found
to be responsible for Parkinson's disease. It is reported that some
people having Italian ancestry suffer from autosomal dominant
Parkinson's disease due to mutation of this gene. There is,
however, limitation in diagnosing Parkinson's disease even with use
of this gene. Therefore, what has been adopted at present as a
diagnosis for Parkinson's disease is merely a clinical approach
based on neurodegenerative symptoms such as resting tremor,
rigidity, akinesia, and disturbance of the righting reflux, and a
levodopa-responsive or dopaminergic compound (agonist) has been
administered as a symptomatic treatment. So far no drastic therapy
has been performed for treating Parkinson's disease.
BRIEF SUMMARY OF THE INVENTION
[0007] The present invention has been made in view of the above. An
object of this invention is to provide an isolated DNA or gene or
gene fragment that is responsible for Parkinson's disease and is
useful in diagnosing and treating the disease etc.; a recombinant
vector; a protein or polypeptide; a monoclonal antibody or
polyclonal antibody; a primer or probe or immobilized nucleic acid
or DNA chip; and an oligonucleotide, or the like.
[0008] The isolated DNA or gene according to this invention that
has overcome the above problems residing in the prior art is:
[0009] {circle over (1)} An isolated DNA or gene: comprising a
full-length base sequence according to the sequence of SEQ ID. Nos.
1 or 3 (SEQ ID. No. 3 does not include a base portion 636 to 719
(corresponding to exon 5 which is described later) of SEQ ID. No.
1, namely, a variant thereof according to alternative splicing), or
a partial sequence thereof, or a base sequence hybridizable thereto
or hybridizable with a complementary strand thereof, and being
associated with Parkinson's disease.
[0010] Further, the inventive DNA or gene may include a DNA or gene
or gene fragment having the following features {circle over (2)} to
{circle over (8)}, in addition to {circle over (1)}.
[0011] {circle over (2)} An isolated DNA or gene: comprising the
base sequence of {circle over (1)}, or the full-length base
sequence thereof, or the base sequence partially thereof, and the
isolated DNA or gene whose gene defect is responsible for
Parkinson's disease, or comprising a base sequence hybridizable
thereto or hybridizable with a complementary strand thereof.
[0012] {circle over (3)} An isolated DNA or gene comprising the
base sequence of {circle over (1)} or {circle over (2)}, the
isolated DNA or gene being variant thereof by alternative splicing,
and being associated with Parkinson's disease, or the isolated DNA
or gene comprising a base sequence hybridizable thereto or
hybridizable with a complementary strand thereof.
[0013] {circle over (4)} A gene comprising the base sequence of any
one of {circle over (1)} to {circle over (3)} whose gene product
encodes a protein having a substantially equivalent function to a
protein comprising amino acids 1 to 465 of SEQ ID. No. 2 or to a
protein comprising amino acids 1 to 437 of SEQ ID. No. 4.
[0014] {circle over (5)} An isolated DNA or gene comprising a gene
where an exonic deletion, a nonsense base substitute, a missense
base substitute, a base deletion, a base addition, a base
insertion, a splicing abnormality and/or a frameshift with respect
to the base sequence has occurred in any one of {circle over (1)}
to {circle over (4)}; or comprising a base sequence hybridizable
thereto or hybridizable with a complementary strand thereof, and
the isolated DNA or gene being associated with Parkinson's
disease.
[0015] {circle over (6)} An isolated DNA or a gene, or a gene
fragment comprising a partial base sequence of the DNA or the gene
of any one of claims {circle over (1)} to {circle over (5)}, or an
isolated DNA or a gene or a gene fragment comprising a base
sequence hybridizable thereto or hybridizable with a complementary
strand thereof.
[0016] {circle over (7)} A gene encoding a protein (a) or (b)
comprising:
[0017] (a) the protein comprising amino acids 1 to 465 of SEQ ID.
No. 2;
[0018] (b) the protein in which one or more amino acid(s) of the
amino acid sequence is or are deleted, substituted, or added, and
the protein being associated with Parkinson's disease.
[0019] {circle over (8)} A gene encoding a protein (c) or (d):
[0020] (c) the protein comprising amino acids 1 to 437 of SEQ ID.
No. 4;
[0021] (d) the protein in which one or more amino acid(s) of the
amino acid sequence is or are deleted, substituted, or added, and
the protein being associated with Parkinson's disease.
[0022] The full-length base SEQ ID. No. 1 is such that eleven
introns are intervened among twelve exons on the genome; and
encodes a protein having 1 to 465 amino acid sequence in a part
(102 to 1496) of the base sequence. The base sequence of the intron
in a boundary region between the exon and the intron has the
following arrangement:
[0023] the intron intervening between exon 1 and exon 2 has a base
sequence shown in SEQ ID. No. 9 adjacent to the 3' end of the exon
1, and has a base sequence shown in SEQ ID. No. 10 adjacent to the
5' end of the exon 2;
[0024] the intron intervening between exon 2 and exon 3 has a base
sequence shown in SEQ ID. No. 11 adjacent to the 3' end of the exon
2, and has a base sequence shown in SEQ ID. No. 12 adjacent to the
5' end of the exon 3;
[0025] the intron intervening between exon 3 and exon 4 has a base
sequence shown in SEQ ID. No. 13 adjacent to the 3' end of the exon
3, and has a base sequence shown in SEQ ID. No. 14 adjacent to the
5' end of the exon 4;
[0026] the intron intervening between exon 4 and exon 5 has a base
sequence shown in SEQ ID. No. 15 adjacent to the 3' end of the exon
4, and has a base sequence shown in SEQ ID. No. 16 adjacent to the
5' end of the exon 5;
[0027] the intron intervening between exon 5 and exon 6 has a base
sequence shown in SEQ ID. No. 17 adjacent to the 3' end of the exon
5, and has a base sequence shown in SEQ ID. No. 18 adjacent to the
5' end of the exon 6;
[0028] the intron intervening between exon 6 and exon 7 has a base
sequence shown in SEQ ID. No. 19 adjacent to the 3' end of the exon
6, and has a base sequence shown in SEQ ID. No. 20 adjacent to the
5' end of the exon 7;
[0029] the intron intervening between exon 7 and exon 8 has a base
sequence shown in SEQ ID. No. 21 adjacent to the 3' end of the exon
7, and has a base sequence shown in SEQ ID. No. 22 adjacent to the
5' end of the exon 8;
[0030] the intron intervening between exon 8 and exon 9 has a base
sequence shown in SEQ ID. No. 23 adjacent to the 3' end of the exon
8, and has a base sequence shown in SEQ ID. No. 24 adjacent to the
5' end of the exon 9;
[0031] the intron intervening between exon 9 and exon 10 has a base
sequence shown in SEQ ID. No. 25 adjacent to the 3' end of the exon
9, and has a base sequence shown in SEQ ID. No. 26 adjacent to the
5' end of the exon 10;
[0032] the intron intervening between exon 10 and exon 11 has a
base sequence shown in SEQ ID. No. 27 adjacent to the 3' end of the
exon 10, and has a base sequence shown in SEQ ID. No. 28 adjacent
to the 5' end of the exon 11; and
[0033] the intron intervening between exon 11 and exon 12 has a
base sequence shown in SEQ ID. No. 29 adjacent to the 3' end of the
exon 11, and has a base sequence shown in SEQ ID. No. 30 adjacent
to the 5' end of the exon 12.
[0034] In addition, a recombinant vector comprising the DNA
fragment or the gene of any one of {circle over (1)} to {circle
over (8)} may be included in the scope of this invention.
[0035] The protein which has overcome the above problem is (i) a
protein comprising amino acids 1 to 465 of SEQ ID. No. 2; or (ii) a
protein comprising amino acids 1 to 437 of SEQ ID. No. 4, the
protein being associated with Parkinson's disease; or a protein
having a substantially equivalent function thereto.
[0036] More specifically, the protein or polypeptide according to
this invention may embrace the following aspects (ii) to
(viii).
[0037] (ii) A protein expressed by the gene of any one of {circle
over (1)} to {circle over (4)}, the protein being associated with
Parkinson's disease, or having an identical function thereto or a
substantially equivalent function thereto.
[0038] (iii) A protein comprising an amino acid sequence translated
by the gene of {circle over (5)}, the protein being associated with
Parkinson's disease, or having an identical function thereto or a
substantially equivalent function thereto.
[0039] (iv) A protein comprising the amino acid sequence of (iii)
in which an amino acid is substituted, deleted, or added at least
at one position, and the protein being associated with Parkinson's
disease.
[0040] (v) A protein comprising the amino acid sequence of any one
of (ii) to (iv) comprising: a ubiquitin-like 1 to 72 amino acid
sequence partially included in SEQ ID. No. 2; and a
zinc-finger-protein-like 418 to 449 amino acid sequence partially
included in SEQ ID. No. 2.
[0041] (vi) A protein (a) or (b):
[0042] (a) the protein comprising amino acids 1 to 465 of SEQ ID.
No. 2;
[0043] (b) the protein in which one or more amino acid(s) of the
amino acid sequence is or are deleted, substituted, or added, and
the protein being associated with Parkinson's disease.
[0044] (vii) A protein (c) or (d):
[0045] (c) the protein comprising amino acids 1 to 437 of SEQ ID.
No. 4;
[0046] (d) the protein in which one or more amino acid(s) of the
amino acid sequence is or are deleted, substituted, or added, and
the protein being associated with Parkinson's disease.
[0047] (viii) A polypeptide or a protein consisting of a partial
fragment of the amino acid sequence of any one of (i) (vii), or
comprising the partial fragment thereof, or the full-length amino
acid sequence thereof.
[0048] In addition, a monoclonal antibody or a polyclonal antibody
against the protein of any one of (i) to (viii) may be included in
the scope of this invention.
[0049] Further, a primer, or a probe, or an immobilized nucleic
acid, or a DNA chip according to this invention may preferably be
used for the following purposes (I) to (IV):
[0050] (I) for use in detecting a base sequence, a genetic
mutation, a deletion, and/or an expression amount of the DNA or the
gene of any one of {circle over (1)} to {circle over (8)}, or for
use in concentration thereof;
[0051] (II) for use in detecting a base sequence, a genetic
mutation, a deletion, and/or an expression amount of RNA which is
subjected to transcription and subjected to processing from the DNA
or the gene of any one of {circle over (1)} to {circle over (8)},
or for use in concentration thereof;
[0052] (III) for use in detecting a base sequence, a genetic
mutation, and/or a deletion of the exon of SEQ ID. No. 1 or No. 3,
or for use in haplotyping a locus thereof; or
[0053] (IV) for use in detecting a base sequence, a genetic
mutation, and/or a deletion of the aforementioned intron, or for
use in haplotyping a locus thereof.
[0054] Specifically, at least one of the fourteen sets of primers
or probes shown in the following (1) to (14) can be used.
[0055] (1) A primer or a probe for use in detecting a base sequence
of the intron adjacent to the exon 1 of the gene being associated
with Parkinson's disease of {circle over (1)}, or a locus thereof,
the primer or probe comprising the following base sequence:
[0056] a base sequence of SEQ ID. No. 31 in the 5'-3' direction of
SEQ ID. No. 1 on the genome, and
[0057] a base sequence of SEQ ID. No. 32 in the 5'-3' direction on
a complementary strand of SEQ ID. No. 1 on the genome.
[0058] (2) A primer or a probe for use in detecting a base sequence
of an intron adjacent to the exon 2 of the gene being associated
with Parkinson's disease of {circle over (1)}, or a locus thereof,
the primer or the probe comprising the following base sequence:
[0059] a base sequence of SEQ ID. No. 33 in the 5'-3' direction of
SEQ ID. No. 1 on the genome, and
[0060] a base sequence of SEQ ID. No. 34 in the 5'-3' direction on
a complementary strand of SEQ ID. No. 1 on the genome.
[0061] (3) A primer or a probe for use in detecting a base sequence
of an intron adjacent to the exon 3 of the gene being associated
with Parkinson's disease of {circle over (1)}, or a locus thereof,
the primer or the probe comprising the following base sequence:
[0062] a base sequence of SEQ ID. No. 35 in the 5'-3' direction of
SEQ ID. No. 1 on the genome, and
[0063] a base sequence of SEQ ID. No. 36 in the 5'-3' direction on
a complementary strand of SEQ ID. No. 1 on the genome.
[0064] (4) A primer or a probe for use in detecting a base sequence
of the intron adjacent to the exon 4 of the gene being associated
with Parkinson's disease of {circle over (1)}, or a locus thereof,
the primer or probe comprising the following base sequence:
[0065] a base sequence of SEQ ID. No. 37 in the 5'-3' direction of
SEQ ID. No. 1 on the genome, and
[0066] a base sequence of SEQ ID. No. 38 in the 5'-3' direction on
a complementary strand of SEQ ID. No. 1 on the genome.
[0067] (5) A primer or a probe for use in detecting a base sequence
of an intron adjacent to the exon 4 of the gene being associated
with Parkinson's disease of {circle over (1)}, or a locus thereof,
the primer or the probe comprising the following base sequence:
[0068] a base sequence of SEQ ID. No. 39 in the 5'-3' direction of
SEQ ID. No. 1 on the genome, and
[0069] a base sequence of SEQ ID. No. 40 in the 5'-3' direction on
a complementary strand of SEQ ID. No. 1 on the genome.
[0070] (6) A primer or a probe for use in detecting a base sequence
of an intron adjacent to the exon 5 of the gene being associated
with Parkinson's disease of {circle over (1)}, or a locus thereof,
the primer or the probe comprising the following base sequence:
[0071] a base sequence of SEQ ID. No. 41 in the 5'-3' direction of
SEQ ID. No. 1 on the genome, and
[0072] a base sequence of SEQ ID. No. 42 in the 5'-3' direction on
a complementary strand of SEQ ID. No. 1 on the genome.
[0073] (7) A primer or a probe for use in detecting a base sequence
of the intron adjacent to the exon 6 of the gene being associated
with Parkinson's disease of {circle over (1)}, or a locus thereof,
the primer or probe comprising the following base sequence:
[0074] a base sequence of SEQ ID. No. 43 in the 5'-3' direction of
SEQ ID. No. 1 on the genome, and
[0075] a base sequence of SEQ ID. No. 44 in the 5'-3' direction on
a complementary strand of SEQ ID. No. 1 on the genome.
[0076] (8) A primer or a probe for use in detecting a base sequence
of an intron adjacent to the exon 7 of the gene being associated
with Parkinson's disease of {circle over (1)}, or a locus thereof,
the primer or the probe comprising the following base sequence:
[0077] a base sequence of SEQ ID. No. 45 in the 5'-3' direction of
SEQ ID. No. 1 on the genome, and
[0078] a base sequence of SEQ ID. No. 46 in the 5'-3' direction on
a complementary strand of SEQ ID. No. 1 on the genome.
[0079] (9) A primer or a probe for use in detecting a base sequence
of an intron adjacent to the exon 7 of the gene being associated
with Parkinson's disease of {circle over (1)}, or a locus thereof,
the primer or the probe comprising the following base sequence:
[0080] a base sequence of SEQ ID. No. 47 in the 5'-3' direction of
SEQ ID. No. 1 on the genome, and
[0081] a base sequence of SEQ ID. No. 48 in the 5'-3' direction on
a complementary strand of SEQ ID. No. 1 on the genome.
[0082] (10) A primer or a probe for use in detecting a base
sequence of the intron adjacent to the exon 8 of the gene being
associated with Parkinson's disease of {circle over (1)}, or a
locus thereof, the primer or probe comprising the following base
sequence:
[0083] a base sequence of SEQ ID. No. 49 in the 5'-3' direction of
SEQ ID. No. 1 on the genome, and
[0084] a base sequence of SEQ ID. No. 50 in the 5'-3' direction on
a complementary strand of SEQ ID. No. 1 on the genome.
[0085] (11) A primer or a probe for use in detecting a base
sequence of an intron adjacent to the exon 9 of the gene being
associated with Parkinson's disease of {circle over (1)}, or a
locus thereof, the primer or the probe comprising the following
base sequence:
[0086] a base sequence of SEQ ID. No. 51 in the 5'-3' direction of
SEQ ID. No. 1 on the genome, and
[0087] a base sequence of SEQ ID. No. 52 in the 5'-3' direction on
a complementary strand of SEQ ID. No. 1 on the genome.
[0088] (12) A primer or a probe for use in detecting a base
sequence of an intron adjacent to the exon 10 of the gene being
associated with Parkinson's disease of {circle over (1)}, or a
locus thereof, the primer or the probe comprising the following
base sequence:
[0089] a base sequence of SEQ ID. No. 53 in the 5'-3' direction of
SEQ ID. No. 1 on the genome, and
[0090] a base sequence of SEQ ID. No. 54 in the 5'-3' direction on
a complementary strand of SEQ ID. No. 1 on the genome.
[0091] (13) A primer or a probe for use in detecting a base
sequence of the intron adjacent to the exon 11 of the gene being
associated with Parkinson's disease of {circle over (1)}, or a
locus thereof, the primer or probe comprising the following base
sequence:
[0092] a base sequence of SEQ ID. No. 55 in the 5'-3' direction of
SEQ ID. No. 1 on the genome, and
[0093] a base sequence of SEQ ID. No. 56 in the 5'-3' direction on
a complementary strand of SEQ ID. No. 1 on the genome.
[0094] (14) A primer or a probe for use in detecting a base
sequence of an intron adjacent to the exon 12 of the gene being
associated with Parkinson's disease of {circle over (1)}, or a
locus thereof, the primer or the probe comprising the following
base sequence:
[0095] a base sequence of SEQ ID. No. 57 in the 5'-3' direction of
SEQ ID. No. 1 on the genome, and
[0096] a base sequence of SEQ ID. No. 58 in the 5'-3' direction on
a complementary strand of SEQ ID. No. 1 on the genome.
[0097] Further, the present invention may include the following
oligonucleotide, or an oligonucleotide analog, or a modified
product thereof as shown in (a) to (c):
[0098] (a) the one which comprises a partial sequence of the base
sequence of any one of {circle over (1)} to {circle over (8)}, or
which is hybridizable with the base sequence of any one of {circle
over (1)} to {circle over (8)}.
[0099] (b) the one for use in amplifying the fill-length base
sequence or the partial base sequence of any one of {circle over
(1)} to {circle over (8)}, or the oligonucleotide for use in
amplifying partially the full-length base sequence or the partial
base sequence of any one of {circle over (1)} to {circle over (8)},
according to PCR method using a human RNA as a template, PCR method
or RT-PCR method using a human cDNA as a template.
[0100] (c) the oligonucleotide for use in amplifying the base
sequence comprising the exon in the sequence ID. No. 1 or No. 3 and
the aforementioned intron which is adjacent to the exon according
to PCR method, or the oligonucleotide for use in amplifying a part
of the base sequence according to PCR method.
BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES
[0101] The file of this patent contains at least one drawing
executed in color. Copies of this patent with color drawing(s) will
be provided by the Patent and Trademark Office upon request and
payment of the necessary fee.
[0102] FIG. 1 is a diagram showing the pedigree of family members
including Parkinson's disease patient in Example 1.
[0103] FIG. 2 is a diagram showing results of haplotyping according
to PCR analysis with respect to the family members including
Parkinson's disease patient in FIG. 1.
[0104] FIG. 3 is a diagram showing presence or absence of gene
deletion in the family members including Parkinson's disease
patient in FIG. 1.
[0105] FIG. 4 is a diagram showing an alignment of six cDNA
fragments and one full-length gene isolated in Example 3.
[0106] FIG. 5 is a diagram showing amino acid sequence homology
between the N-terminus of this inventive gene and ubiquitin. The
aligned sequences include Parkin (amino acids 1-76 of SEQ ID NO:2),
Human Ubiquitin (SEQ ID NO:60), Yeast Ubiquitin (SEQ ID NO:61) and
Soybean Ubiquitin (SEQ ID NO:62).
[0107] FIG. 6 is a diagram showing the result of gel
electrophoresis of EcoRI digest of genomic DNA fragments with
respect to the family members including Parkinson's disease patient
in FIG. 1.
[0108] FIG. 7 is a diagram showing the pedigree of family members
including Parkinson's disease patients in Example 7.
[0109] FIG. 8 is a diagram showing presence or absence of gene
deletion with respect to the family members including Parkinson's
disease patient in FIG. 7.
[0110] FIG. 9 is a diagram showing the sequence of a cDNA fragment
(SEQ ID NO:63) of the Parkinson's disease patient in FIG. 7,
obtained by Example 7.
[0111] FIGs. 10A-10C are diagram showing mRNAs of the inventive
gene that are expressed in various human tissues.
[0112] FIGS. 11A-11B are diagrams showing mRNAs of the inventive
gene that are expressed in various human tissues
[0113] FIG. 12 is a diagram showing the pedigree of family members
including Parkinson's disease patients in Example 9.
[0114] FIG. 13 is a diagram showing presence or absence of gene
deletion in the family members including Parkinson's disease
patient in FIG. 12.
[0115] FIG. 14 is a diagram showing the result of gel
electrophoresis of EcoRI digest of genomic DNA fragments with
respect to the family members including Parkinson's disease patient
in FIG. 12.
[0116] FIG. 15 is a diagram showing the pedigree of family members
including Parkinson's disease patients in Example 10.
[0117] FIG. 16 is a diagram showing presence or absence of gene
deletion with respect to the family members including Parkinson's
disease patient in FIG. 15.
[0118] FIG. 17 is a diagram showing the pedigree of family members
including Parkinson's disease patients in Example 11.
[0119] FIG. 18 is a diagram showing presence or absence of gene
deletion with respect to the family members in FIG. 17.
[0120] FIG. 19 is a diagram showing the pedigree of family members
including Parkinson's disease patients in Example 12.
[0121] FIG. 20 is a diagram showing presence or absence of gene
deletion with respect to the family members including Parkinson's
disease patient in FIG. 19.
[0122] FIG. 21 is a diagram showing the pedigree of family members
including Parkinson's disease patients in Example 13.
[0123] FIG. 22 is a diagram showing presence or absence of gene
deletion with respect to the family members including Parkinson's
disease patient in FIG. 21.
[0124] FIG. 23 is a diagram showing the pedigree of family members
including Parkinson's disease patient in Example 14.
[0125] FIG. 24 is a chromatogram showing the result of direct
sequencing of PCR products from exon 5 of a wild allele (SEQ ID
NO:64) and a mutant allele (SEQ ID NO:65).
[0126] FIG. 25 is a diagram showing DNA (SEQ ID NO:66) and amino
acid (SEQ ID NO:67) sequences of a wild-type (W) parkin gene, and
predicted DNA (SEQ ID NO:68) and amino acid (SEQ ID NO:69)
sequences of a mutant (M) parkin molecule having one-base
deletion.
[0127] FIG. 26 is a diagram showing the result of N1aIV restriction
site analysis of PCR products in Example 14.
[0128] FIGS. 27A-27I are photomicrographs showing the result of
immunohistochemical staining of the inventive gene that are
expressed in brain sections.
[0129] FIGS. 28A-28I are photomicrographs showing the result of
immunostaining of the inventive gene that are expressed in brain
sections, polyubiquitin, and .alpha.-synuclein.
[0130] FIG. 29 is a diagram showing the result of immunoblotting
the whole homogenates of the frontal lobe of control subjects,
Parkinson's disease patients, and AR-JP patients.
[0131] FIG. 30 is a diagram showing the result of immunoblotting
the subcellular fractions of the frontal lobe tissue of a control
subject.
[0132] FIG. 31 is a diagram showing the result of immunoblotting
the whole homogenates of the SN, putamen, and frontal lobe of
control subjects, Parkinson's disease patients, and AR-JP
patient.
[0133] FIGS. 32A-32C are diagrams showing the result of gel
electrophoresis of PCR products from exon 4 and exon 10.
[0134] FIG. 33 is a graph obtained by hydropathy plot of the amino
acid sequence of this inventive protein.
[0135] FIG. 34 is a diagram showing that replacement of 366 Arg to
Trp changes a helix portion to .beta.-sheet structure.
DETAILED DESCRIPTION OF THE INVENTION
[0136] Parkinson's disease or Parkinsonism is considered to be
initiated by genetic predisposition and environmental factors.
Elucidating individual factors is an urgent matter on fundamental
understanding and treatment of Parkinson's disease and Parkinsonism
in their onset stage. The inventors of this invention have studied
to find out a gene responsible for onset of Parkinson's disease. As
a result of their study, it was found that the region of chromosome
6q25.2-q27, more specifically, a 17-cM region between two
chromosome markers DS437 and D6S264 has a strong linkage with
juvenile Parkinsonism, which is one of the family of Parkinson's
diseases (Matsumine et al., Am. J. Hum. Genet. 60(1997)588-596).
The inventors have succeeded in isolating the gene responsible for
Parkinson's disease by conducting the below-mentioned Examples with
respect to the juvenile Parkinson's disease patients and thus
accomplished the present invention.
[0137] Hereinafter, the present invention is described in detail
with reference to the process of experiments that have contributed
to the finding of the inventive gene. It should be appreciated that
the following examples are illustrative and not restrictive, and
all changes that fall within metes and bounds of the claims, or
equivalence of such metes and bounds are therefore intended to be
embraced by the claims.
EXAMPLE 1
Chromosomal Deletion Region in Juvenile Parkinson's Disease
Patient
[0138] FIG. 1 shows the pedigree of family members including
Parkinson's disease patient in Example 1. In FIG. 1, an open square
represents an unaffected male, an open circle represents an
unaffected female, and a filled circle represents the affected
female. The circle or square with slash represents the deceased
member. Although the parents and brothers of the patient are not
affected, the patient had Parkinson-like symptom from her teens and
diagnosed as Parkinson's disease. The symptom has been gradually
progressing.
[0139] Haplotyping according to PCR method was performed using
D6S305 which is one of the markers of chromosome 6 with respect to
the genomic DNA of subjects marked with an asterisk in FIG. 1
(patient and two unaffected members). The result is shown in FIG.
2.
[0140] As shown in FIG. 2, D6S305 was amplified from DNA template
of the parents and brother of the patient, however, D6S305 was not
amplified from DNA template of the patient. It was verified
accordingly that the patient has deletion of D6S305 which is one of
the chromosomal markers.
EXAMPLE 2
Screening of Genomic Fragment Including D6S305 and Exon
Trapping
[0141] Since Example 1 verified that the patient has deletion of
the genomic DNA corresponding to marker D6S305, there is a
possibility that a gene responsible for Parkinson's disease may
exist on the genomic DNA. To verify the possibility, PCR screening
was performed to isolate normal human genomic library consisting of
96,000 genomic fragment (the Keio human BAC library) using a set of
amplimers having a sequence of part of marker D6S305. As a result
of screening, two clones, genomic fragments KB761D4 and KB430C4
each of which has an insert size of about 110 kb were isolated.
[0142] Next, exon trapping was performed to isolate exon fragment
of the gene existing on the genomic fragments using exon trapping
system (provided by GIBCO/BRL) according to the manufacturer's
instruction manual. As a result, the isolated exon was J-17 only
despite the fact that the two genomic fragments each had a
relatively large size of about 110 kb.
[0143] Subsequently, base sequence of the intron adjacent to exon
J-17 was determined based on the base sequence of exon J-17 by
using a PCR primer to amplify exon J-17 itself (J-17 Iner), and BAC
KB761D4 as template. Then, two sets of PCR primer (J-17 outer) were
prepared to amplify the fragment including J-17 based on the
thus-determined sequence of the intron. In this way, PCR
amplification analysis was performed for the genomic DNA of the
subjects shown in Example 1 (patient and the unaffected parents and
brother). The result of analysis is shown in FIG. 3. It should be
noted that FIG. 3 shows the result of Example 6 as well as the
result of Example 2.
[0144] As shown in FIG. 3, no PCR product was detected from the
genomic DNA of the patient (lane 3) whereas PCR product was
detected from the normal genomic DNA of the father (lane 1), mother
(lane 2) and brother (lane 4). This suggests that the patient has
at least chromosomal deletion corresponding to exon J-17 of the
inventive gene.
EXAMPLE 3
Screening of Inventive Gene from Normal Human cDNA Library
[0145] Next, screening of the inventive gene was performed using
human cDNA library to isolate cDNAs which cover the full-length of
the gene including J-17 together with full-length of its
translation sequence. Specifically, cDNA libraries of normal human
fetal brain and skeletal muscle were purchased from Clontech. J-17
fragment, which is part of exon of the inventive gene and was
isolated in Example 2, was used as an initial probe, and insertion
DNA fragment of positive clones isolated by initial screening using
the initial probe were used as probes for secondary screening. As a
result, seven cDNA clones [HFB1, HFB3, HFB4, HFB5, SKM1, SKM3, and
SKM8] shown in FIG. 4 were isolated. The insertion DNA fragments of
positive clones were amplified with two set of vector-specific
primer (F10inner: 5'-AGCCTGGTTAAGTCCAAGCTG-3' (SEQ ID NO:5) and
R10inner: 5'-GAAGGTCCCATTTTTCGTTTTC-3' (SEQ ID NO:6)).
[0146] The thus amplified positive DNA fragment was sequenced
directly according to primer walking method. Cycle sequencing was
performed using the above-mentioned primers and a commercial kit
[ABI PRISM labeling kits (manufactured by Perkin-Elmer)] and ABI
model 377DNA sequencer (manufactured by Applied Biosystems)
according to the manufacture's instruction manual.
[0147] As a result, it was found that seven cDNAs had an piled
relationship as shown in FIG. 4. The longest base sequence SKM8 has
2960bp which includes a full-length of translation sequence which
encodes a protein containing 1395bp (nt102 to nt1496 or to nt1499
including stop codon), and 465 amino acids.
[0148] Also, it was found that four cDNA clones (HFB3, HFB4, SKM1,
and SKM3) of seven cDNA lost 84bp from nt636 to nt719. This
implicates that there are at least two ways of splicing when mature
mRNA grows from this inventive gene on genome by splicing.
[0149] Further, it was verified that the N-terminal portion
(ranging from methionine-1 to arginine-72) of the protein
consisting of 1 to 465 amino acid sequence which is encoded by the
inventive gene has a moderate homology (content of the same amino
acid: 33%) with ubiquitin as shown in FIG. 5.
[0150] Ubiquitin is known as a significant substance which removes
a protein that has no longer been necessary in a cell, and
involvement with various neurodegenerative diseases has also been
pointed out. For instance, it has been known that paired helical
filaments (PHFs) in Alzheimer's disease and Lewy bodies in
Parkinson's disease are stained by an anti-polyubiquitin antibody.
The mechanism is considered to act as follows. Ubiquitin is
conjugated with various proteins and forms multi-ubiquitin chain by
repeated conjugations, and induces the proteasome pathway, finally
to be metabolized.
[0151] Lysine residue-48 is known to be an essential element for
ubiquitin-conjugate. Since lysine exists at position 48 in the
protein of the current invention, and the amino acid sequence at
the vicinity of the target region (for instance, positions 44 to 48
and position 51) conforms with that of ubiquitin, it is suggested
that the protein has ubiquitin-like function. Further, recent
studies found some of conjugated proteins contain ubiquitin-like
portion at the N-terminal portion thereof. The latter finding
implies that the ubiquitin-like portion acts as a molecular
chaperone.
[0152] Although homology with ubiquitin is observed at the
N-terminal portion of this inventive protein as described above,
homology with ubiquitin is seldom observed with respect to amino
acid sequence at position 73 and thereafter. As another feature,
the protein of this invention has amino acid sequence at the
vicinity of the C-terminal portion (positions 418 to 449)
containing a large number of cysteine residues:
Cys-X2-Cys-X9-Cys-X1-His-X2-Cys-X4-Cys-X4-Cys-X2-Cys (SEQ ID NO:7).
This sequence is extremely similar to that of a ring-finger motif
(Cys-X2-Cys-X(9-399)-Cys-X(1-3)-His-X(2-3)-Cys-X2-Cys-X(4-48)-Cys-X2-Cys)
(SEQ ID NO:8), a kind of sequence of a zinc-binding motif in a
zinc-finger protein (a protein conjugated with zinc, and deeply
involved in growth, differentiation, and generation of a cell).
Accordingly, it is presumed that the protein of this invention is
one of novel zinc-finger proteins.
EXAMPLE 4
Screening of the Inventive Genomic Gene from Genome Library
[0153] As mentioned above, only exon (J-17) was found in two
positive genomic clones (KB761D4 and KB430C4) which were obtained
in Example 2. In this Example, by the purpose of obtaining a
genomic fragment containing other exon(s), BAC clone screening was
performed by hybridization of DNA from a genome library consisting
of 95,232 clones (Keio human BAC library), using SKM8 clone which
has the largest size among the positive clones obtained from the
aforementioned cDNA library, as a probe. As a result, 24 new
positive clones were obtained.
[0154] In addition, a PCR primer for amplifying exon 1, which
corresponds to the N-terminal portion of the cDNA base sequence was
prepared, screening of BAC library according to PCR amplification
was performed, and another new positive clone was obtained.
[0155] Identification of each exon and sequencing of intron
adjacent to each exon were performed according to Primer walking
method (BEE procedure) using the above-obtained twenty five clones.
As a result of analysis, exon 1 to 3, 5, 6, and 8-12 were mapped to
either one of the twenty five BAC clones. Also, it was verified
that J-17 corresponds to exon 7. BAC clones having genomic sequence
including exon 4 were not, however, found in the twenty five BAC
clones.
[0156] Another PCR primer to amplify exon 4 was prepared, and two
new positive clones were obtained by PCR screening using a genome
library supplied by Genome Systems Inc. Sequencing of each exon and
intron adjacent to each exon was performed according to the
aforementioned primer walking method with use of twenty seven
BAC-DNA clones as template. The primers used in primer walking
method were appropriately prepared based on cDNA sequence. BAC
clones corresponding to the respective primers were separated
according to oligonucleotide colony hybridization using primer
itself as a probe. DNA sequencer was used for their sequencing.
[0157] As a result, the alignment of exon and intron of this
inventive gene was made clear. It was verified that the gene of
this invention has a very large spanning over 500 kb and consists
of twelve exons intervening very large eleven introns. The intron
sequence in the boundary region between exon and intron was
described as above. Table 1 shows the whole base sequences in
exon-intron boundaries.
1TABLE 1 Intron - exon boundaries of Parkin gene Exon Intron Exon
Exon 1ACCATGATAG gtacgtgggt TGTTTGTCAG Exon 2 (SEQ ID NO: 9) . . .
. ccttggtcag (SEQ ID NO: 10) Exon 2GACTGTGCAG gtgagtctcc AATTGTGACC
Exon 3 (SEQ ID NO: 11) . . . . tcccaaacag (SEQ ID NO: 12) Exon
3GGAAGTCCAG gtaattggaa CAGGTAGATC Exon 4 (SEQ ID NO: 13) . . . .
tcttctccag (SEQ ID NO: 14) Exon 4CTTGACCCAG gtaaggaaat GGTCCATCTT
Exon 5 (SEQ ID NO: 15) . . . . tttcccaaag (SEQ ID NO: 16) Exon
5GACTAGTGCA gtaagtacct GAATTTTTCT Exon 6 (SEQ ID NO: 17) . . . .
tttctttcag (SEQ ID NO: 18) Exon 6CAGACGTCAG gtaaggatct GAGCCCCGTC
Exon 7 (SEQ ID NO: 19) . . . . ctctctgcag (SEQ ID NO: 20) Exon
7CCTTGTGTGG gtaagtctag CTGGCTGTCC Exon 8 (SEQ ID NO: 21) . . . .
tttccaacag (SEQ ID NO: 22) Exon 8AGAAGAGCAG gtgagtgagc TACAACCGGT
Exon 9 (SEQ ID NO: 23) . . . . ggttttgcag (SEQ ID NO: 24) Exon
9GGGCTGTGGG gtgagtactg TTTGCCTTCT Exon 10 (SEQ ID NO: 25) . . . .
tcttttgcag (SEQ ID NO: 26) Exon 10AACTACTCAG gtacagaatg GCCTACAGAG
Exon 11 (SEQ ID NO: 27) . . . . gtttccccag (SEQ ID NO: 28) Exon
11GAAAAAAATG gtgagtctgt GAGGCTGCAT Exon 12 (SEQ ID NO: 29) . . . .
cccccaacag (SEQ ID NO: 30)
EXAMPLE 5
Determination of Base Sequence of Exonic Part on Genomic DNA
[0158] Next, the base sequence of exon part obtained in Example 4
was amplified to verify whether the base sequence of the part
conforms with that of the corresponding part of cDNA. Specifically,
fourteen sets of primers were prepared based on flanking intron
sequence at the 5'-terminus and 3'-terminus of each exon (part of
the primer was prepared based on partial sequence of exon), and PCR
amplification was performed with use of DNAs which have been
prepared by the standard procedure with use of normal human
peripheral blood leukocytes as template. For reference, Table 2
shows the base sequences of the fourteen primer sets used in this
Example.
2TABLE 2 Primer sequences and sizes of expected PCR products
product size Exon Primer Forward (5'-3") Reverse (5'-3') (bp) 1 Ex
1 GCGCGGCTGGCGCCGCTGCGCGCA GCGGCGCAGAGAGGCTGTAC 112 (SEQ ID NO: 31)
(SEQ ID NO: 32) 2 Ex 2 ATGTTGCTATCACCATTTAAGGG
AGATTGGCAGCGCAGGCGGCATG 308 (SEQ ID NO: 33) (SEQ ID NO: 34) 3 Ex 3
ACATGTCACTTTTGCTTCCCT AGGCCATGCTCCATGCAGACTGC 427 (SEQ ID NO: 35)
(SEQ ID NO: 36) 4 Ex 4 inner AGGTAGATCAATCTACAACAGCT
CTGGGTCAAGGTGAGCGTTGCCTGC 121 (SEQ ID NO: 37) (SEQ ID NO: 38) 4 Ex
4 outer ACAAGCTTTTAAAGAGTTTCTTGT AGGCAATGTGTTAGTACACA 261 (SEQ ID
NO: 39) (SEQ ID NO: 40) 5 Ex 5 ACATGTCTTAAGGAGTACATTT
TCTCTAATTTCCTGGCAAACAGTG 227 (SEQ ID NO: 41) (SEQ ID NO: 42) 6 Ex 6
AGAGATTGTTTACTGTGGAAACA GAGTGATGCTATTTTTAGATCCT 268 (SEQ ID NO: 43)
(SEQ ID NO: 44) 7 J-17 inner GAGCCCCGTCCTGGTTTTCC
CCACACAAGGCAGGGAGTAGCCAA 137 (SEQ ID NO: 45) (SEQ ID NO: 46) 7 J-17
outer TGCCTTTCCACACTGACAGGTACT TCTGTTCTTCATTAGCATTAGAGA 239 (SEQ ID
NO: 47) (SEQ ID NO: 48) 8 Ex 8 TGATAGTCATAACTGTGTGTAAG
ACTGTCTCATTAGCGTCTATCTT 206 (SEQ ID NO: 49) (SEQ ID NO: 50) 9 Ex 9
GGGTGAAATTTGCAGTCAGT AATATAATCCCAGCCCATGTGCA 278 (SEQ ID NO: 51)
(SEQ ID NO: 52) 10 Ex 10 ATTGCCAAATGCAACCTAATGTC
TTGGAGGAATGAGTAGGGCATT 165 (SEQ ID NO: 53) (SEQ ID NO: 54) 11 Ex 11
ACAGGGAACATAAACTCTGATCC CAACACACCAGGCACCTTCAGA 303 (SEQ ID NO: 55)
(SEQ ID NO: 56) 12 Ex 12 GTTTGGGAATGCGTGTTTT
AGAATTAGAAAATGAAGGTAGACA 255 (SEQ ID NO: 57) (SEQ ID NO: 58)
[0159] The above PCR amplification was carried out in the following
manner. In this Example, 10 ml-reactions were prepared, each of
which contained 100 ng DNA, 1.times.PCR buffer [50 mM Tris-HCl (pH
9.2 at 25.degree. C.), 14 mM (NH4)2SO4, 1.75 mM MgCl2], 350 .mu.M
each dNTP, 0.5 .mu.M each primer and 0.35 U Expand Long Taq
polymerase (Boerhinger Manheim). PCR conditions were at 94.degree.
C for 30 sec., 50-53.degree. C. for 30 sec., 68.degree. C. for 30
sec. to 1 min. and repeated 35 cycles.
[0160] Base sequence of each DNA fragment that has been amplified
by the above PCR was determined using appropriate PCR primers and a
commercial kit. The result of sequencing is shown in Table 2.
[0161] As shown in Table 2, it was verified that the base sequence
amplified according to the PCR using the aforementioned primers
conforms with that of the corresponding part of cDNA.
EXAMPLE 6
Partial Deletion of Inventive Gene in Juvenile Parkinson's Disease
Patient
[0162] (Case 1)
[0163] In this Example, abnormality of the inventive gene was
examined using the juvenile Parkinson's disease patient and family
members in Example 1.
[0164] Specifically, genomic DNAs were prepared from the leukocytes
of the subjects, and PCR amplification was carried out using the
genomic DNAs as template and primer sets consisting of forward
(5'-3') and reverse (5'-3') of exon 2, exon 3, J-17 inner, J-17
outer, and exon 8 among the primer sets listed in Table 2. The
result of analysis is shown in FIG. 3.
[0165] As seen from FIG. 3, in the case where the genomic DNAs of
father (lane 1), mother (lane 2), and brother (lane 4) of the
Parkinson's disease patient were used as template, the sequence
corresponding to each exon was amplified. This result verifies that
the genomic DNAs of these family members do not have deletion or
significant mutation. On the other hand, in the case where the
genomic DNA of the Parkinson's disease patient (lane 3) was used as
template, no amplification of the base sequence of the genomic DNA
corresponding to exons 3, 4, 5, 6, 7 was found. This result
clarified that the genomic gene of the patient has a deletion of
long base sequence corresponding to exons 3 to 7.
[0166] Furthermore, the genomic DNAs of the subjects were digested
with EcoRI, electrophoresed, and blotted onto nylon membrane by
Southern blot analysis, and P.sup.32-labeling of SKM8 cDNA probe
was performed by Southern blot hybridization. As a result, as shown
in FIG. 6., whereas at least eight EcoRI fragments were found in
the parents and brother of the patient, only four fragments were
found in the patient (in FIG. 6, asterisk denotes the four EcoRI
fragment that were not detected in the patient). This result also
verifies that the genomic gene of the patient has deletion or
mutation at a certain part thereof.
EXAMPLE 7
Partial Deletion of Inventive Gene in Juvenile Parkinson's Disease
Patient
[0167] (Case 2)
[0168] As can be seen from Example 6, it is obvious that juvenile
Parkinson's disease patients have deletion or the like in the
inventive gene. The above example strongly implicates that deletion
or the like of the inventive gene is responsible for juvenile
Parkinson's disease. To further verify this, similar experiments
were conducted with respect to another unrelated family members to
those in Example 6.
[0169] Specifically, genome analysis was carried out with respect
to the family members including juvenile Parkinson's disease
patients of the pedigree in FIG. 7. Whereas two siblings out of six
are unaffected, the other four siblings are all juvenile
Parkinson's disease patients. PCR analysis was performed in
accordance with the procedure in Example 5 using primers
corresponding to respective exons with use of the genomic DNAs of
the members marked with asterisk (namely, unaffected mother, two
unaffected brothers, and two affected sisters) as template. The
result of analysis is shown in FIG. 8.
[0170] As can be seen from FIG. 8, whereas both of the genomic DNAs
of the two patients (lanes 2 and 3) in this Example show deletion
of exon 4, none of the genomic DNAs of the other subjects
(unaffected mother (lane 1) and two unaffected sisters (lane 5 and
6)) have deletion.
[0171] Furthermore, mRNA was extracted from the brain tissue of one
of the patients according to the standard AGPC procedure. Total 1
mg of mRNA was primed at 50.degree. C. for 30 min. using TitanTM
one tube RT-PCR System kit (Boehringer Manheim), and the reaction
mixture was directly used for PCR with forward primer (nt 351 to nt
371 of SEQ ID No. 1) 5'-GGAGGCGACGACCCCAGAAAC-3' and reverse primer
(nt 963 to nt 983 of SEQ ID. No. 1) 5'-GGGACAGCCAGCCACACAAGG-3'
(SEQ ID NO:70). PCR was performed at 94.degree. C. for 30 sec.,
56.degree. C. for 30 sec., 68.degree. C. for 1 min. and repeated 45
cycles. cDNA sequence of PCR products obtained by the above
procedure was analyzed, and the result of analysis is shown in FIG.
9.
[0172] As seen from FIG. 9, mRNA of the patient shows complete
deletion of exon 4, exon 3 is contiguous to exon 5 directly by
skipping exon 4. Consequently, it was verified that the juvenile
Parkinson's disease patients of two unrelated family members have
deletion of exon in the inventive gene and that deletion of the
inventive gene is responsible for juvenile Parkinson's disease.
EXAMPLE 8
Inventive Gene mRNA Expression in Various Tissues
[0173] In this Example, Northern blot analysis was carried out
using the genomic fragment J-17 in order to examine how widely mRNA
[Poly(A)+] of the inventive gene is expressed in various human
tissues.
[0174] Specifically, Northern blots of various human tissues were
purchased from Clontech, and northern blotting was carried out
according to the provided instruction manual with use of J-17
corresponding to exon 4 of the inventive gene, as a probe. The
result of analysis is shown in FIGS. 10 to 11. It should be noted
that tissues in FIG. 10A are, from left to right in the order, are
spleen, thymus, prostate, testis, ovary, small intestine, colon,
and peripheral blood leukocyte; those in FIG. 10B are, from left to
right in the order, heart, brain, placenta, lung, liver, skeletal
muscle, kidney, and pancreas; those in FIG. 10C are, from left to
right in the order, stomach, thyroid, spinal cord, lymph node,
trachea, adrenal gland, and bone marrow; those in FIG. 11A are,
from left to right in the order, cerebellum, cerebral cortex,
medulla, spinal cord, occipital pole, frontal lobe, temporal lobe,
and putamen; and those in FIG. 11B are, from left to right in the
order, amygdala, caudate nucleus, corpus callosum, hippocampus,
whole brain, substantia nigra, subthalamic nucleus, and
thalamus.
[0175] The results of FIGS. 10 to 11 show that mRNA was
particularly richly expressed in the tissue of brain, heart, testis
and skeletal muscle although mRNA of 4.5 kb including poly A-tail
was detected in all the tissues examined in this Example. It was
further verified that expression was particularly remarkable in the
cerebral cortex and frontal lobe although the expression was
detected in every section of the brain.
EXAMPLE 9
Partial Deletion of Inventive Gene in Juvenile Parkinson's Disease
Patient
[0176] (Case 3)
[0177] FIG. 12 shows the pedigree of family members including
Parkinson's disease patients in Example 9. In this Example, genomic
DNAs were prepared from leukocytes of the subjects marked with
numerals 1 to 7 in FIG. 12 (namely, unaffected parents, three
unaffected sisters, and two affected brothers) and used as
template. PCR amplification was performed using oligonucleotide
primer pairs shown in Table 3.
3TABLE 3 Primer sequences and sizes of PCR products product size
Exon Primer Forward (5'-3") Reverse (5'-3') (bp) 1 Ex 1
GCGCGGCTGGCGCCGCTGCGCGCA GCGGCGCAGAGAGGCTGTAC 112 (SEQ ID NO: 31)
(SEQ ID NO: 32) 2 Ex 2 ATGTTGCTATCACCATTTAAGGG
AGATTGGCAGCGCAGGCGGCATG 308 (SEQ ID NO: 33) (SEQ ID NO: 34) 3 Ex 3
ACATGTCACTTTTGCTTCCCT AGGCCATGCTCCATGCAGACTGC 427 (SEQ ID NO: 35)
(SEQ ID NO: 36) 4 Ex 4 ACAAGCTTTTAAAGAGTTTCTTGT
AGGCAATGTGTTAGTACACA 261 (SEQ ID NO: 39) (SEQ ID NO: 40) 5 Ex 5
ACATGTCTTAAGGAGTACATTT TCTCTAATTTCCTGGCAAACAGTG 227 (SEQ ID NO: 41)
(SEQ ID NO: 42) 6 Ex 6 AGAGATTGTTTACTGTGGAAACA
GAGTGATGCTATTTTTAGATCCT 268 (SEQ ID NO: 43) (SEQ ID NO: 44) 7 Ex 7
TGCCTTTCCACACTGACAGGTACT TCTGTTCTTCATTAGCATTAGAGA 239 (SEQ ID NO:
47) (SEQ ID NO: 48) 8 Ex 8 TGATAGTCATAACTGTGTGTAAG
ACTGTCTCATTAGCGTCTATCTT 206 (SEQ ID NO: 49) (SEQ ID NO: 50) 9 Ex 9
GGGTGAAATTTGCAGTCAGT AATATAATCCCAGCCCATGTGCA 278 (SEQ ID NO: 51)
(SEQ ID NO: 52) 10 Ex 10 ATTGCCAAATGCAACCTMTGTC
TTGGAGGAATGAGTAGGGCATT 165 (SEQ ID NO: 59) (SEQ ID NO: 54) 11 Ex 11
ACAGGGAACATAAACTCTGATCC CAACACACCAGGCACCTTCAGA 303 (SEQ ID NO: 55)
(SEQ ID NO: 56) 12 Ex 12 GTTTGGGAATGCGTGTTTT
AGAATTAGAAAATGAAGGTAGACA 255 (SEQ ID NO: 57) (SEQ ID NO: 58)
[0178] All the reactions were carried out according to the
following procedure. Prepared was a 25 .mu.l reaction mixture
containing 50 mM KCl, 10 mM Tris (pH 8.3), 1.5 mM MgCl2, 0.02%
gelatin with primers, 10 nmol of each dNTP, and 2.5 units of
AmpliTaq Gold DNA polymerase (Perkin-Elmer Applied Biosystems
Division). Initial denaturation at 94.degree. C. for 10 min. was
followed by 40 cycles of 94.degree. C. for 30 sec., 55.degree. C.
for 30 sec., and 72.degree. C. for 45 sec., and then a final
extension at 72.degree. C. for 10 min. The PCR products were
visualized on ethidium bromide-stained 2% agarose gels and the
presence or absence of the target exon(s) was detected. The result
is shown in FIG. 13.
[0179] As seen in FIG. 13, in the case where DNA of the two
Parkinson's disease patients (lanes 6, 7) was used as template, no
amplification of the regions corresponding to exon 5 was
detected.
[0180] A further experiment was carried out. The genomic DNA of the
Parkinson's disease patient (marked with numeral 6 in FIG. 12) and
of his father was digested with EcoRI, electrophoresed, and blotted
onto nylon membrane by Southern blotting. Then, P.sup.32-labeling
of SKM8 DNA probe was subjected to Southern blot hybridization. The
result of this analysis is shown in FIG. 14. As can be seen from
FIG. 14, whereas at least eight EcoRI fragments were detected in
the father, only seven EcoRI fragments were detected in the patient
(in FIG. 14, asterisk mark denotes the undetected EcoRI fragment).
This analysis verifies that the genomic gene of the Parkinson's
disease patient has deletion or mutation in a specific regions of
the inventive gene.
EXAMPLE 10
Partial Deletion of Inventive Gene in Juvenile Parkinson's Disease
Patient
[0181] (Case 4)
[0182] Another experiment was carried out in accordance with the
procedure in Example 9 except that unrelated another family members
to those in Example 9 were examined. Specifically, genomic analysis
was carried out for the family members including juvenile
Parkinson's disease patients of the pedigree shown in FIG. 15. Four
sisters out of seven are unaffected, but the other three sisters
have juvenile Parkinson's disease. PCR analysis was performed in
accordance with the procedure in Example 9 using primers
corresponding to respective exons with use of the genomic DNAs of
the subjects marked with numerals 1 to 6 in FIG. 15, as template.
The result of analysis is shown in FIG. 16.
[0183] As shown in FIG. 16, in the case where the DNAs of the
Parkinson's disease patients (lane 3 and 6) were used as template,
no amplification was found with respect to the base sequence of the
regions corresponding to exon 3.
EXAMPLE 11
Partial Deletion of Inventive Gene in Juvenile Parkinson's Disease
Patient
[0184] (Case 5)
[0185] Another experiment was performed in accordance with the
procedure in Example 9 except that unrelated family members to
those in above Examples were examined. In this Example, genomic
analysis was performed for the family members including juvenile
Parkinson's disease patients of the pedigree shown in FIG. 17.
Three siblings out of five are unaffected, but the other two
siblings are juvenile Parkinson's disease patients. PCR analysis
was performed in accordance with the procedure in Example 9 using
primers corresponding to respective exons with use of the genomic
DNAs of the subjects marked with numerals 1 to 3 in FIG. 17, as
template. The result of analysis is shown in FIG. 18.
[0186] As shown in FIG. 18, in the case where the DNA of the
Parkinson's disease patient (lane 1) was used as template, no
amplification was observed with respect to the base sequence of the
regions corresponding to exon 4.
EXAMPLE 12
Partial Deletion of Inventive Gene in Juvenile Parkinson's Disease
Patient
[0187] (Case 6)
[0188] A further experiment was conducted in accordance with the
procedure in Example 9 except that unrelated family members to
those in above Examples were examined. Specifically, genomic
analysis was performed for the family members including juvenile
Parkinson's disease patients of the pedigree shown in FIG. 19.
Parents and six siblings out of eight are unaffected, but the other
two siblings are juvenile Parkinson's disease patients. PCR
analysis was performed in accordance with the procedure in Example
9 using primers corresponding to respective exons with use of the
genomic DNAs of the subjects marked with numerals 1 to 8 in FIG.
19, as template. The result of analysis is shown in FIG. 20.
[0189] As shown in FIG. 20, in the case where the DNA of the
Parkinson's disease patients (lane 4 and 8) were used as template,
no amplification was observed with respect to the base sequence of
the DNAs corresponding to exon 3 and exon 4.
EXAMPLE 13
Partial Deletion of Inventive Gene in Juvenile Parkinson's Disease
Patient
[0190] (Case 7)
[0191] Another experiment was conducted in accordance with the
procedure in Example 9 except that unrelated family members to
those in the above Examples were examined. Specifically, genomic
analysis was performed for the family members including juvenile
Parkinson's disease patients of the pedigree shown in FIG. 21.
Parents and one brother out of five siblings are unaffected, but
the other four siblings are juvenile Parkinson's disease patients.
PCR analysis was performed in accordance with the procedure in
Example 9 using primers corresponding to respective exons with use
of the genomic DNAs of the subjects marked with numerals 1 to 6 in
FIG. 21, as template. The result of analysis is shown in FIG.
22.
[0192] As shown in FIG. 22, in the case where the DNA of the
Parkinson's disease patients (lane 2 to 6) were used as template,
no amplification was observed with respect to the base sequence of
the regions corresponding to exon 5.
EXAMPLE 14
Identification of Homozygous One-base Deletion in Exon 5
[0193] Screening was performed to determine deletion, insertion or
point mutation according to direct sequencing PCR for one patient
each from the family members of pedigree shown in FIG. 23 etc. PCR
was performed with chimera primers that were specific to
oligonucleotide primer sequences and had the sequences of the
standard sequencing primers (M13 universal and reverse primers) at
their 5'-ends, respectively. Excess primers and dNTPs were removed
from the PCR products with an Ultrafree-MC centrifugal filter
(Millipore). The purified PCR products were sequenced by the
dideoxy chain termination method with an Applied Biosystems 373A
DNA sequencer.
[0194] As a result of screening, one-base deletion in exon 5 was
identified among the patients (see FIG. 24). In FIG. 24, the upper
section (N) represents the result of direct sequencing of the PCR
products from exon 5 of a wild allele, and the lower section (M)
represents the result of direct sequencing of the PCR products from
exon 5 of a mutant allele.
[0195] More specifically, the one-base deletion removed one
guanosine from the sequence -GGT- (codon 179), causing a frameshift
that resulted in an intermediate stop codon at amino acid position
187. The nucleotide and predicted amino acid sequences are shown in
FIG. 25. In FIG. 25, "Normal" section shows DNA and amino acid
sequences of a wild-type allele, and "Mutant" section shows DNA and
amino acid sequences of a mutant allele with one-base deletion,
respectively. This one-base deletion was not detected in the normal
subjects.
[0196] Next, to verify the one-base deletion in the patient (marked
with numeral 3) of the pedigree shown in FIG. 23 and to identify
the genotypes of her parents (marked with numerals 1 and 2) and her
unaffected sister (marked with numeral 4), N1aIV restriction site
analysis was performed. In this analysis, exon 5 of the subjects
was amplified by primer pairs in accordance with the aforementioned
procedure, and their PCR products were digested with N1aIV (New
England Biolabs Inc., Massachusetts). The PCR products were
electrophoresed on 3% (2% Agarose/1% NuSieve Agarose) gel and
visualized with ethidium bromide. The result is shown in FIG. 26.
In FIG. 26, lane 1 shows the sequence of father, lane 2 shows that
of mother, lane 3 shows that of the patient, and lane 4 shows that
of the unaffected sister, respectively.
[0197] The wild-type allele can be detected as an N1aIV site in
exon 5, and digestion with N1aIV produced two fragments (159 bp and
68 bp). On the other hand, the mutant allele having one-base
deletion showed a single fragment of 227 bp. This restriction site
analysis verified that the patient is mutant homozygote due to
one-base deletion in exon 5 whereas her parents are wild-type
heterozygotes, and her unaffected sister is wild-type homozygote.
These results are. consistent with the mode of autosomal recessive
mode of inheritance.
EXAMPLE 15
Immunohistochemical and Immunofluorescence Analysis of Inventive
Gene in Juvenile Parkinson's Disease Patients
[0198] In order to elucidate the molecular mechanism of substantia
nigra (SN) caused by mutation of the inventive gene (hereinafter,
sometimes referred to as "Parkin"), localization of the protein of
this invention in the brains of patients with autosomal recessive
juvenile Parkinsonism (AR-JP), sporadic Parkinson's disease (PD)
and normal control subjects was examined by using antibodies
against the protein of this invention.
[0199] More specifically, cases of fifteen PD patients, three AR-JP
patients, and eight control subjects were studied. Among AR-JP
patients, case 1 and case 2 are sisters, and they had a deletion of
exon 4 in the inventive gene, resulting in a truncated protein of
143 amino acids due to a stop codon generated by the frameshift 6
amino acids after codon 138. Case 3 of AR-JP patient was a 52
year-old female patient and she had a deletion of exon 3 which
causes a premature termination at amino acid 96 due to the
frameshift after amino acid 58.
[0200] Two kinds of rabbit polyclonal antibodies (M-73 and M-74),
rabbit polyclonal antibody against .alpha.-synuclein, and mouse
monoclonal antibody against polyubiquitin were used respectively in
this Example.
[0201] First of all, immunohistochemical staining was conducted
according to the following procedure. Formalin-fixed
paraffin-embedded sections of the midbrain, frontal lobe cortex,
and putamen of the subjects were treated with anti-Parkin M-74,
anti-.alpha.-synuclein, or anti-polyubiquitin antibodies after
appropriate dilution by a standard avidin-biotin complex method
using 3',3'-diaminobenzidine for visualization.
Double-immunofluorescence was performed with rabbit anti-Parkin
antibody M74 and mouse anti-polyubiquitine monoclonal antibody, and
subsequent incubation with FITC-conjugated goat anti-rabbit IgG
(Dako, Carpinteria, Calif.), biotinylated goat anti-mouse IgG
(Sigma, St. Louis, Mo.) and avidin-rhodamine (Sigma). Signal was
observed under a fluorescent confocal microscope MRC-1024 (Bio-Rad,
Richmond, Calif.). The results of observation are shown in FIGS. 27
and 28.
[0202] FIG. 27 are photomicrographs of immunohistochemical staining
with anti-Parkin antibody M-74 in the brain sections, wherein 27A
to 27C show the result of a PD patient (case 2), 27D to 27F show
that of a control subject (case 1), and 27G to 27I show that of a
AR-JP patient (case 1). More specifically, the photomicrographs
27A, 27D, 27G show the melanin-containing neurons in the SN, 27B,
27E, 27H show the putamen, and 27C, 27F, 27I show the frontal lobe
cortex. In the photomicrographs, the point of arrow indicates
neuron, the root thereof indicates neuromelanin, and the unit
length of bar thereof is 50 .mu.m.
[0203] As can be seen from FIG. 27, melanin-containing neurons in
the SN (including locus coeruleus) were most intensely stained in
the PD patient and the control subject, but not in the AR-JP
patient (FIGS. 27A, 27D, 27G). Further, in these melanin-containing
neurons of the SN, cytoplasm and granular structure as well as
neuronal processes were homogeneously stained. In contrast, no
staining was seen in the nuclei. Some weak staining was observed in
glial cells (FIGS. 27A to 27F). Neurons in the putamen and frontal
lobe cortex from the PD patient and the control subject were weakly
stained in the cytoplasm and perinuclear structures (FIGS. 27B,
27C, 27E, 27F).
[0204] FIG. 28 is photomicrographs of immunohistochemical staining
with this inventive gene polyubiquitin, and .alpha.-synuclein in
the brain sections, wherein 28A to 28C show the melanin-containing
neuron in the SN of a PD patient (case 1) double-stained with
anti-Parkin antibody M74 (green: A) and monoclonal
anti-polyubiquitin antibody (red: B), 28D to 28F show midbrain
cross-sections from the PD patient (case 1) stained with
anti-.alpha.-synuclein antibody, and 28G to 28I show midbrain
cross-sections from the PD patient (case 1) stained with
anti-Parkin antibody M-74. In the photomicrographs of FIG. 28, the
root of arrow indicates Lewy body, and the unit length of bar
thereof is 50 .mu.m.
[0205] As a result of double immunofluorescence, the anti-Parkin
and anti-polyubiquitin antibodies in the Lewy body of
melanin-containing neurons of the SN were stained (FIGS. 28A to
28C). As a result of immunostaining of cross-sections of midbrain,
co-localization of this inventive gene and .alpha.-synuclein in
some of Lewy bodies of the PD patient (FIGS. 28D, 28E, 28G, 28H)
was observed. No such staining was observed in the brain tissues of
the AR-JP patients (data not shown).
[0206] The above observation results verify that whereas the
protein of this invention was observed in the brains of the
sporadic PD patients and control subjects, this protein was not
observed in the brains of the AR-JP patients. Also, the protein of
this invention was found in Lewy body of the PD patients.
EXAMPLE 16
Immunoblotting of Inventive Gene in Juvenile Parkinson's Disease
patients
[0207] Followed by Example 15, in this Example, immunoblotting was
carried out with respect to the inventive gene existing in the
brains of AR-JP patients, PD patients and control subjects with use
of antibodies against the protein of this invention.
[0208] Specifically, tissue blocks of frontal lobe cortex,
substantia nigra and putamen of the subjects were homogenized with
a Potter-Elvehjem homogenizer in an isotonic sucrose solution (10
mM Tris-HCl pH 7.4, 0.32 M sucrose, 1 mM Zn-acetate, 15 .mu.g/ml
leupeptin, 5 .mu.g/ml p-amidinophenylmethanesulfonyl fluoride
hydrochloride (APMSF) and 50 ng/ml pepstatin). The homogenate was
processed for 4 step-differential centrifugation to obtain the
following fractions; Nuclear fraction (pellets after 600.times.g
for 10 min.), mitochondrial fraction (pellets after 7,000.times.g
for 10 min.), microsomal fraction (pellets after 100,000.times.g
for 1 hr.) and cytosolic fraction (supernatant after
900,000.times.g for 1 hr.). The 900,000.times.g pellet was
resuspended in TES buffer containing 0.25 M sucrose and layered
over a step-gradient of sucrose (0.25 M, 0.86 M and 1.3 M) and
centrifuged in an SW28 rotor at 28,000 rpm for 1 hr. at 4.degree.
C. After lipid on the top layer was aspirated, the interface
between 0.5 M and 0.86 M sucrose layers was collected as the Golgi
fraction.
[0209] Proteins in these various fractions were separated on a 10%
sodium dodecyl sulfate-polyacrylamide gel electrophoresis and
transferred to PVDF membrane blots (Bio-Rad). The blots were soaked
in Tris-buffered saline containing 0.05% Tween 20 and 5% bovine
serum albumin (10 mM Tris-HCl, pH 7.6 and 150 mM NaCl) at
52.degree. C. for 1 hr., probed with various antibodies such as
anti-Parkin antibody M-74 in the blocking solution at 4.degree. C.
overnight, then washed with Tris-buffered saline containing 0.05%
Tween 20. Anti-.beta.-tubulin antibody (Amersham Life Science,
Arlington, Ill.) was used as an internal control and
anti-.gamma.-adaptin antibody (Sigma) was used as a Golgi marker.
Finally, blots were treated with peroxidase-conjugated goat
anti-rabbit IgG (Dako) and anti-mouse IgG (Dako) at a room
temperature for 1 hr. Then the reaction products were visualized
using a chemi-luminescence reagent (Amersham, Buckinghamshire,
UK).
[0210] The results of immunoblotting are shown in FIGS. 29 to 31.
FIGS. 29 to 31 are microphotographs showing the results of
immunoblotting the protein of this invention in various
homogenates. FIG. 29 shows the result of whole homogenates of the
frontal lobe of three control subjects (cases 1 to 3), three PD
patients (cases 1 to 3) and two AR-JP patients (cases 1 and 2),
wherein the left side gels are size markers, and .beta.-tubulin is
an internal marker. FIG. 30 shows the result of subcellular
fractions of the frontal lobe tissue of the control subject (case
1) wherein nuclear, mitochondria, microsome, cytosol and Golgi are
shown from left to right in the order, and .gamma.-adaptin is a
Golgi marker. FIG. 31 shows the result of whole homogenates of the
SN, putamen and frontal lobe of the two control subjects (cases 1
and 2) and the two PD patients (cases 2 and 3).
[0211] As a result, the protein of this invention (in FIGs denoted
as "Parkin") of 52 kDa was detected in the whole homogenates of the
frontal lobe cortex from the PD patients and the control subjects
but not in those of the AR-JP patients (see FIG. 29). A second
protein band of 41 kDa, possibly a processed form of Parkin
protein, was found in the PD patients. Similar results were
obtained using another antibody M-73 (data not shown).
[0212] After subcellular fractionation of the frontal lobe cortex
homogenates of the control subject, majority of the inventive
protein was found in the cytosol and Golgi fractions, and a minute
amount of the inventive protein was found in the microsomal
fraction (see FIG. 30).
[0213] Further, immunoblotting analysis of the homogenates of the
SN, putamen and frontal lobe cortex from the control subjects and
PD patients revealed that the inventive protein is more abundant in
the SN as compared to the other parts of the brain (see FIG. 31).
The inventive protein in the SN Of the Parkinson's disease patients
was obviously reduced in agreement with the loss of nigral neurons
in the PD patients.
[0214] The above results verified that the protein of this
invention was not detected in any brain section of the AR-JP
patients and that this protein exists in the melanin-containing
neurons in the SN.
EXAMPLE 17
Polymorphism of the Parkin Gene in Sporadic Parkinson's Disease
(PD) Patients and Control Subjects
[0215] In this Example, polymorphism frequency in PD was
investigated. Specifically, hereditary polymorphism was analyzed
according to the following procedure with respect to the subjects
consisting of 160 PD patients and 160 control subjects without
neurodegenerative disorders. In this Example, patients with the age
of onset below 40 years old were excluded. The average age of onset
was 55.4.+-.10.7. None of the PD patients had family history of PD,
nor diurnal fluctuations of symptoms. The age of the control
subjects was from 40 to 98 years old.
[0216] Human genomic DNA was extracted from the peripheral
leukocytes of the subjects. Samples were either used immediately or
stored at -20.degree. C. until analyzed. Exons 4 and 10 of the
Parkin gene were amplified by PCR using two primer pairs (exon 4:
forward primer, 5'-acaagcttttaaagagtttcttgt-3' (SEQ ID NO:39),
reverse primer, 5'-aggcaatgtgttagtacaca-3'(SEQ ID NO:40), exon 10:
forward primer, 5'-attgccaaatgcaacctaatgtc-3' (SEQ ID NO:53),
reverse primer, 5'-ttggaggaatgagtagggcatt-3' (SEQ ID NO:54)).
[0217] Polymorphism that replaces Ser at amino acid position 167 to
Asn (S167N) (replacement of G to A) was found in exon 4.
Polymorphisms that replace Arg at amino acid position 366 to Trp
(R366W) (replacement of C to T) and Val at amino position 380 to
Leu (V380L) (replacement of G to C) respectively were found in exon
10. Alleles of the polymorphisms S167N, R366W, and V380L were
respectively identified by digestion with A1wNI, NciI, BsP1286I.
Whereas both the S167N and R366W wild alleles created restriction
sites for A1wNI and NciI, respectively, the V380L mutant allele
created a restriction site for Bsp 1286I, thus identifying wild
allele and mutant allele.
[0218] More specifically, the PCR products were electrophoresed on
3% agarose gel and then visualized with ethidium bromide. As a
result, as shown in FIG. 32, a band spanning 50 bp/131 bp was found
by digestion with A1wNI [see FIG. 32 A)], a band spanning 68 bp/97
bp was found by digestion with NciI [see FIG. 32 B)], and a band
spanning 57 bp/108 bp was found by digestion with Bsp1286I [see
FIG. 32 C)].
[0219] More specifically, the PCR conditions for exon 4 were as
follows. The initial denaturation was performed at 94.degree. C.
for 10 min., followed by 40 cycles of denaturation at 94.degree. C.
for 30 sec., annealing at 53.degree. C. for 1 min, and extension at
72.degree. C. for 1 min., with a final extension at 72.degree. C.
for 10 min. The PCR conditions for exon 10 were as follows. The
initial denaturation was performed at 94.degree. C. for 10 min.,
followed by 40 cycles of denaturation at 94.degree. C. for 30 sec.,
annealing at 55.degree. C. for 30 sec., and extension at 72.degree.
C. for 45 sec., with a final extension at 72.degree. C. for 10
min.
[0220] Subsequently, frequencies of wild-type homozygotes (w/w),
wild/mutant heterozygotes (w/m), and mutant homozygotes (m/m) were
examined.
[0221] Tables 4 and 5 show the wild allele and genotype frequencies
of the polymorphism S167N. Expected values in Table 5 were
calculated according to the Hardy-Weinberg equilibrium.
4TABLE 4 Control (%) PD (%) Total (%) No. of Subjects 160 160 320
No. of Chromosomes 320 320 640 Allele G 180 (56.3%) 181 (56.6%) 361
(56.4%) Allele A 140 (43.7%) 139 (43.4%) 279 (43.6%) .chi..sup.2 =
0.006, d.f. = 0.936.sup.a Remarks a: No significant difference in
allele frequencies between the PD patients and the control. Note:
Expected values were calculated according to the Hardy-Weinberg
equilibrium.
[0222]
5 TABLE 5 Control (%) PD (%) Observed Expected Observed Expected
Total (%) GG 58 (36.3%) 51 59 (36.9%) 51 117 (36.6%) GA 64 (40.0%)
79 63 (39.4%) 79 127 (39.7%) AA 38 (23.7%) 30 38 (23.7%) 30 76
(23.7%) .chi..sup.2 = 2.97, d.f. = 2, p = 0.227.sup.b .chi..sup.2 =
3.33, d.f. = 2, p = 0.189.sup.c .chi..sup.2 = 0.016, d.f. = 2, p =
0.992.sup.d Remarks: .sup.bThe expected frequencies versus observed
frequencies of the genotype in the control. .sup.cThe expected
versus observed frequencies of the genotype in the PD patients.
.sup.dNo significant difference in the genotype distribution
between the PD patients and the controls.
[0223] The above results show that there is no significant
difference between the PD patients and the control subjects with
respect to allele and genotype frequency. Further, the frequencies
of both -167Ser homozygote and -167Ser/Asn heterozygotes did not
differ significantly between the two groups.
[0224] Further, Table 5 shows that the observed frequencies of
three genotypes did not significantly differ between the expected
frequencies of the control subjects and those of the patients.
Computer analysis verified that this replacement did not cause
changes in the secondary structure of the gene products.
[0225] Table 6 shows the allele and genotype frequencies of the
polymorphism V380L.
6TABLE 6 Control (%) PD (%) Total (%) No. of Subjects 160 160 320
No. of Chromosomes 320 320 640 Allele G 309 (96.6%) 314 (98.1%) 623
(97.3%) Allele C 11 (3.4%) 6 (1.9%) 17 (2.7%) .chi..sup.2 = 1.51,
d.f. = 1, p = 0.219.sup.a Remarks a: No significant difference in
allele frequencies between the PD patients and the controls.
[0226] As shown in Table 6, there was no significant difference in
allele frequencies of the polymorphism V380L between the PD
patients and the controls. Further, it was verified that the
observed frequencies of the polymorphism V380L conformed with the
expected frequencies and that the secondary structure of the gene
product did not change due to this polymorphic mutation.
[0227] Next, Table 7 shows the allele and genotype frequencies of
the polymorphism R366W.
7TABLE 7 Control (%) PD (%) Total (%) No. of Subjects 160 160 320
No. of Chromosomes 320 320 640 Allele C 306 (95.6%) 316 (98.8%) 622
(97.2%) Allele T 14 (4.4%) 4 (1.2%) 18 (2.8%) .chi..sup.2 = 5.72,
d.f. = 1, p = 0.017.sup.a Remarks a: Significant difference in
allele frequencies between the PD patients and the control
subjects. (Fisher's exact probability test, p = 0.014 < 0.02,
Odds ratio = 3.60, 95% Cl: 0.45-6.50).
[0228] Regarding the polymorphism R366W, the expected frequencies
of the three genotypes were exactly same between the PD patients
and the control subjects. However, the allele frequency of R366W
differed significantly between the PD patients and the control
subjects. Specifically, while the allele frequency in the PD
patients was 1.2%, that in the control subjects was 4.4%. This
result shows that the allele frequency in the PD patients
significantly lowered compared to that in the control subjects. The
odd ratio (possession ratio of allele of control subjects to PD
patients) was 3.60.
[0229] FIG. 33 is a graph representing hydropathy index of amino
acid sequence in the polymorphism R366W. (+) and (-) regions in
FIG. 33 represents hydrophobic and hydrophilic regions,
respectively. The point shown by the arrow in FIG. 33 indicates the
change from hydrophilicity to hydrophobicity that is caused by
replacement of -366 Arg with Trp.
[0230] FIG. 34 is a diagram showing a secondary structural change
in the polymorphism R366W. This diagram shows that replacement of
-366 Arg with Trp changes the .alpha.-helix (at position 361 to
376) to the .beta.-sheet structure (at position 360-360).
[0231] This Example shows that although S167N and V380L among the
three polymorphisms did not crucially influence the gene product of
the PD patients, the allele frequency of polymorphism R366W was
extremely low in the PD patients, and the odd ratio was calculated
as 3.60. This result suggests that the allele constitute a
protective factor against PD which inhibits onset of PD.
Exploitation in Industry
[0232] The invention of this application has the above-mentioned
arrangements described in terms of various aspects. The
aforementioned various examples and descriptions not only
identified the gene responsible for Parkinson's disease but also
clarified that partial deletion or mutation etc., of the gene of
this invention induces Parkinson's disease. Accordingly, onset of
Parkinson's disease is easily judged by detecting the presence or
absence of abnormality of the inventive gene, which is very useful
in diagnosing Parkinson's disease at an initial or early stage
thereof.
[0233] So-called "gene therapy" for treating Parkinson's disease
patients with use of the inventive gene is also possible. Further,
recombinant protein obtainable from the inventive gene is useful as
a drug for preventing and/or treating Parkinson's disease. Antibody
(monoclonal antibody and polyclonal antibody) against such a
recombinant protein can be used for diagnosis etc., of Parkinson's
disease. Utilizing such a recombinant protein enables one to
synthesize a pharmaceutically effective agent that is significantly
useful in preventing, treating, and diagnosing Parkinson's disease
etc. Thus the present invention possesses significant usability in
industry because the present invention can contribute to
development of various gene therapies and pharmaceutical
compositions effective in various diseases focusing on Parkinson's
disease and Parkinson-related diseases.
Sequence CWU 1
1
70 1 2960 DNA Homo sapiens CDS (102)..(1496) 1 tccgggagga
ttacccagga gaccgctggt gggaggcgcg gctggcgccg ctgcgcgcat 60
gggcctgttc ctggcccgca gccgccacct acccagtgac c atg ata gtg ttt gtc
116 Met Ile Val Phe Val 1 5 agg ttc aac tcc agc cat ggt ttc cca gtg
gag gtc gat tct gac acc 164 Arg Phe Asn Ser Ser His Gly Phe Pro Val
Glu Val Asp Ser Asp Thr 10 15 20 agc atc ttc cag ctc aag gag gtg
gtt gct aag cga cag ggg gtt ccg 212 Ser Ile Phe Gln Leu Lys Glu Val
Val Ala Lys Arg Gln Gly Val Pro 25 30 35 gct gac cag ttg cgt gtg
att ttc gca ggg aag gag ctg agg aat gac 260 Ala Asp Gln Leu Arg Val
Ile Phe Ala Gly Lys Glu Leu Arg Asn Asp 40 45 50 tgg act gtg cag
aat tgt gac ctg gat cag cag agc att gtt cac att 308 Trp Thr Val Gln
Asn Cys Asp Leu Asp Gln Gln Ser Ile Val His Ile 55 60 65 gtg cag
aga ccg tgg aga aaa ggt caa gaa atg aat gca act gga ggc 356 Val Gln
Arg Pro Trp Arg Lys Gly Gln Glu Met Asn Ala Thr Gly Gly 70 75 80 85
gac gac ccc aga aac gcg gcg gga ggc tgt gag cgg gag ccc cag agc 404
Asp Asp Pro Arg Asn Ala Ala Gly Gly Cys Glu Arg Glu Pro Gln Ser 90
95 100 ttg act cgg gtg gac ctc agc agc tca gtc ctc cca gga gac tct
gtg 452 Leu Thr Arg Val Asp Leu Ser Ser Ser Val Leu Pro Gly Asp Ser
Val 105 110 115 ggg ctg gct gtc att ctg cac act gac agc agg aag gac
tca cca cca 500 Gly Leu Ala Val Ile Leu His Thr Asp Ser Arg Lys Asp
Ser Pro Pro 120 125 130 gct gga agt cca gca ggt aga tca atc tac aac
agc ttt tat gtg tat 548 Ala Gly Ser Pro Ala Gly Arg Ser Ile Tyr Asn
Ser Phe Tyr Val Tyr 135 140 145 tgc aaa ggc ccc tgt caa aga gtg cag
ccg gga aaa ctc agg gta cag 596 Cys Lys Gly Pro Cys Gln Arg Val Gln
Pro Gly Lys Leu Arg Val Gln 150 155 160 165 tgc agc acc tgc agg cag
gca acg ctc acc ttg acc cag ggt cca tct 644 Cys Ser Thr Cys Arg Gln
Ala Thr Leu Thr Leu Thr Gln Gly Pro Ser 170 175 180 tgc tgg gat gat
gtt tta att cca aac cgg atg agt ggt gaa tgc caa 692 Cys Trp Asp Asp
Val Leu Ile Pro Asn Arg Met Ser Gly Glu Cys Gln 185 190 195 tcc cca
cac tgc cct ggg act agt gca gaa ttt ttc ttt aaa tgt gga 740 Ser Pro
His Cys Pro Gly Thr Ser Ala Glu Phe Phe Phe Lys Cys Gly 200 205 210
gca cac ccc acc tct gac aag gaa aca cca gta gct ttg cac ctg atc 788
Ala His Pro Thr Ser Asp Lys Glu Thr Pro Val Ala Leu His Leu Ile 215
220 225 gca aca aat agt cgg aac atc act tgc att acg tgc aca gac gtc
agg 836 Ala Thr Asn Ser Arg Asn Ile Thr Cys Ile Thr Cys Thr Asp Val
Arg 230 235 240 245 agc ccc gtc ctg gtt ttc cag tgc aac tcc cgc cac
gtg att tgc tta 884 Ser Pro Val Leu Val Phe Gln Cys Asn Ser Arg His
Val Ile Cys Leu 250 255 260 gac tgt ttc cac tta tac tgt gtg aca aga
ctc aat gat cgg cag ttt 932 Asp Cys Phe His Leu Tyr Cys Val Thr Arg
Leu Asn Asp Arg Gln Phe 265 270 275 gtt cac gac cct caa ctt ggc tac
tcc ctg cct tgt gtg gct ggc tgt 980 Val His Asp Pro Gln Leu Gly Tyr
Ser Leu Pro Cys Val Ala Gly Cys 280 285 290 ccc aac tcc ttg att aaa
gag ctc cat cac ttc agg att ctg gga gaa 1028 Pro Asn Ser Leu Ile
Lys Glu Leu His His Phe Arg Ile Leu Gly Glu 295 300 305 gag cag tac
aac cgg tac cag cag tat ggt gca gag gag tgt gtc ctg 1076 Glu Gln
Tyr Asn Arg Tyr Gln Gln Tyr Gly Ala Glu Glu Cys Val Leu 310 315 320
325 cag atg ggg ggc gtg tta tgc ccc cgc cct ggc tgt gga gcg ggg ctg
1124 Gln Met Gly Gly Val Leu Cys Pro Arg Pro Gly Cys Gly Ala Gly
Leu 330 335 340 ctg ccg gag cct gac cag agg aaa gtc acc tgc gaa ggg
ggc aat ggc 1172 Leu Pro Glu Pro Asp Gln Arg Lys Val Thr Cys Glu
Gly Gly Asn Gly 345 350 355 ctg ggc tgt ggg ttt gcc ttc tgc cgg gaa
tgt aaa gaa gcg tac cat 1220 Leu Gly Cys Gly Phe Ala Phe Cys Arg
Glu Cys Lys Glu Ala Tyr His 360 365 370 gaa ggg gag tgc agt gcc gta
ttt gaa gcc tca gga aca act act cag 1268 Glu Gly Glu Cys Ser Ala
Val Phe Glu Ala Ser Gly Thr Thr Thr Gln 375 380 385 gcc tac aga gtc
gat gaa aga gcc gcc gag cag gct cgt tgg gaa gca 1316 Ala Tyr Arg
Val Asp Glu Arg Ala Ala Glu Gln Ala Arg Trp Glu Ala 390 395 400 405
gcc tcc aaa gaa acc atc aag aaa acc acc aag ccc tgt ccc cgc tgc
1364 Ala Ser Lys Glu Thr Ile Lys Lys Thr Thr Lys Pro Cys Pro Arg
Cys 410 415 420 cat gta cca gtg gaa aaa aat gga ggc tgc atg cac atg
aag tgt ccg 1412 His Val Pro Val Glu Lys Asn Gly Gly Cys Met His
Met Lys Cys Pro 425 430 435 cag ccc cag tgc agg ctc gag tgg tgc tgg
aac tgt ggc tgc gag tgg 1460 Gln Pro Gln Cys Arg Leu Glu Trp Cys
Trp Asn Cys Gly Cys Glu Trp 440 445 450 aac cgc gtc tgc atg ggg gac
cac tgg ttc gac gtg tagccagggc 1506 Asn Arg Val Cys Met Gly Asp His
Trp Phe Asp Val 455 460 465 ggccgggcgc cccatcgcca catcctgggg
gagcataccc agtgtctacc ttcattttct 1566 aattctcttt tcaaacacac
acacacacgc gcgcgcgcgc acacacactc ttcaagtttt 1626 tttcaaagtc
caactacagc caaattgcag aagaaactcc tggatccctt tcactatgtc 1686
catgaaaaac agcagagtaa aattacagaa gaagctcctg aatccctttc agtttgtcca
1746 cacaagacag cagagccatc tgcgacacca ccaacaggcg ttctcagcct
ccggatgaca 1806 caaataccag agcacagatt caagtgcaat ccatgtatct
gtatgggtca ttctcacctg 1866 aattcgagac aggcagaatc agtagctgga
gagagagttc tcacatttaa tatcctgcct 1926 tttaccttca gtaaacacca
tgaagatgcc attgacaagg tgtttctctg taaaatgaac 1986 tgcagtgggt
tctccaaact agattcatgg ctttaacagt aatgttctta tttaaatttt 2046
cagaaagcat ctattcccaa agaaccccag gcaatagtca aaaacatttg tttatcctta
2106 agaattccat ctatataaat cgcattaatc gaaataccaa ctatgtgtaa
atcaacttgt 2166 cacaaagtga gaaattatga aagttaattt gaatgttgaa
tgtttgaatt acagggaaga 2226 aatcaagtta atgtactttc attccctttc
atgatttgca actttagaaa gaaattgttt 2286 ttctgaaagt atcaccaaaa
aatctatagt ttgattctga gtattcattt tgcaacttgg 2346 agattttgct
aatacatttg gctccactgt aaatttaata gataaagtgc ctataaagga 2406
aacacgttta gaaatgattt caaaatgata ttcaatctta acaaaagtga acattattaa
2466 atcagaatct ttaaagagga gcctttccag aactaccaaa atgaagacac
gcccgactct 2526 ctccatcaga agggtttata cccctttggc acaccctctc
tgtccaatct gcaagtccca 2586 gggagctctg cataccaggg gttccccagg
agagaccttc tcttaggaca gtaaactcac 2646 tagaatattc cttatgttga
catggattgg atttcagttc aatcaaactt tcagcttttt 2706 tttcagccat
tcacaacaca atcaaaagat taacaacact gcatgcggca aaccgcatgc 2766
tcttacccac actacgcaga agagaaagta caaccactat cttttgttct acctgtattg
2826 tctgacttct caggaagatc gtgaacataa ctgagggcat gagtctcact
agcacatgga 2886 ggcccttttg gatttagaga ctgtaaatta ttaaatcggc
aacagggctt ctctttttag 2946 atgtagcact gaaa 2960 2 465 PRT Homo
sapiens 2 Met Ile Val Phe Val Arg Phe Asn Ser Ser His Gly Phe Pro
Val Glu 1 5 10 15 Val Asp Ser Asp Thr Ser Ile Phe Gln Leu Lys Glu
Val Val Ala Lys 20 25 30 Arg Gln Gly Val Pro Ala Asp Gln Leu Arg
Val Ile Phe Ala Gly Lys 35 40 45 Glu Leu Arg Asn Asp Trp Thr Val
Gln Asn Cys Asp Leu Asp Gln Gln 50 55 60 Ser Ile Val His Ile Val
Gln Arg Pro Trp Arg Lys Gly Gln Glu Met 65 70 75 80 Asn Ala Thr Gly
Gly Asp Asp Pro Arg Asn Ala Ala Gly Gly Cys Glu 85 90 95 Arg Glu
Pro Gln Ser Leu Thr Arg Val Asp Leu Ser Ser Ser Val Leu 100 105 110
Pro Gly Asp Ser Val Gly Leu Ala Val Ile Leu His Thr Asp Ser Arg 115
120 125 Lys Asp Ser Pro Pro Ala Gly Ser Pro Ala Gly Arg Ser Ile Tyr
Asn 130 135 140 Ser Phe Tyr Val Tyr Cys Lys Gly Pro Cys Gln Arg Val
Gln Pro Gly 145 150 155 160 Lys Leu Arg Val Gln Cys Ser Thr Cys Arg
Gln Ala Thr Leu Thr Leu 165 170 175 Thr Gln Gly Pro Ser Cys Trp Asp
Asp Val Leu Ile Pro Asn Arg Met 180 185 190 Ser Gly Glu Cys Gln Ser
Pro His Cys Pro Gly Thr Ser Ala Glu Phe 195 200 205 Phe Phe Lys Cys
Gly Ala His Pro Thr Ser Asp Lys Glu Thr Pro Val 210 215 220 Ala Leu
His Leu Ile Ala Thr Asn Ser Arg Asn Ile Thr Cys Ile Thr 225 230 235
240 Cys Thr Asp Val Arg Ser Pro Val Leu Val Phe Gln Cys Asn Ser Arg
245 250 255 His Val Ile Cys Leu Asp Cys Phe His Leu Tyr Cys Val Thr
Arg Leu 260 265 270 Asn Asp Arg Gln Phe Val His Asp Pro Gln Leu Gly
Tyr Ser Leu Pro 275 280 285 Cys Val Ala Gly Cys Pro Asn Ser Leu Ile
Lys Glu Leu His His Phe 290 295 300 Arg Ile Leu Gly Glu Glu Gln Tyr
Asn Arg Tyr Gln Gln Tyr Gly Ala 305 310 315 320 Glu Glu Cys Val Leu
Gln Met Gly Gly Val Leu Cys Pro Arg Pro Gly 325 330 335 Cys Gly Ala
Gly Leu Leu Pro Glu Pro Asp Gln Arg Lys Val Thr Cys 340 345 350 Glu
Gly Gly Asn Gly Leu Gly Cys Gly Phe Ala Phe Cys Arg Glu Cys 355 360
365 Lys Glu Ala Tyr His Glu Gly Glu Cys Ser Ala Val Phe Glu Ala Ser
370 375 380 Gly Thr Thr Thr Gln Ala Tyr Arg Val Asp Glu Arg Ala Ala
Glu Gln 385 390 395 400 Ala Arg Trp Glu Ala Ala Ser Lys Glu Thr Ile
Lys Lys Thr Thr Lys 405 410 415 Pro Cys Pro Arg Cys His Val Pro Val
Glu Lys Asn Gly Gly Cys Met 420 425 430 His Met Lys Cys Pro Gln Pro
Gln Cys Arg Leu Glu Trp Cys Trp Asn 435 440 445 Cys Gly Cys Glu Trp
Asn Arg Val Cys Met Gly Asp His Trp Phe Asp 450 455 460 Val 465 3
2876 DNA Homo sapiens CDS (102)..(1412) 3 tccgggagga ttacccagga
gaccgctggt gggaggcgcg gctggcgccg ctgcgcgcat 60 gggcctgttc
ctggcccgca gccgccacct acccagtgac c atg ata gtg ttt gtc 116 Met Ile
Val Phe Val 1 5 agg ttc aac tcc agc cat ggt ttc cca gtg gag gtc gat
tct gac acc 164 Arg Phe Asn Ser Ser His Gly Phe Pro Val Glu Val Asp
Ser Asp Thr 10 15 20 agc atc ttc cag ctc aag gag gtg gtt gct aag
cga cag ggg gtt ccg 212 Ser Ile Phe Gln Leu Lys Glu Val Val Ala Lys
Arg Gln Gly Val Pro 25 30 35 gct gac cag ttg cgt gtg att ttc gca
ggg aag gag ctg agg aat gac 260 Ala Asp Gln Leu Arg Val Ile Phe Ala
Gly Lys Glu Leu Arg Asn Asp 40 45 50 tgg act gtg cag aat tgt gac
ctg gat cag cag agc att gtt cac att 308 Trp Thr Val Gln Asn Cys Asp
Leu Asp Gln Gln Ser Ile Val His Ile 55 60 65 gtg cag aga ccg tgg
aga aaa ggt caa gaa atg aat gca act gga ggc 356 Val Gln Arg Pro Trp
Arg Lys Gly Gln Glu Met Asn Ala Thr Gly Gly 70 75 80 85 gac gac ccc
aga aac gcg gcg gga ggc tgt gag cgg gag ccc cag agc 404 Asp Asp Pro
Arg Asn Ala Ala Gly Gly Cys Glu Arg Glu Pro Gln Ser 90 95 100 ttg
act cgg gtg gac ctc agc agc tca gtc ctc cca gga gac tct gtg 452 Leu
Thr Arg Val Asp Leu Ser Ser Ser Val Leu Pro Gly Asp Ser Val 105 110
115 ggg ctg gct gtc att ctg cac act gac agc agg aag gac tca cca cca
500 Gly Leu Ala Val Ile Leu His Thr Asp Ser Arg Lys Asp Ser Pro Pro
120 125 130 gct gga agt cca gca ggt aga tca atc tac aac agc ttt tat
gtg tat 548 Ala Gly Ser Pro Ala Gly Arg Ser Ile Tyr Asn Ser Phe Tyr
Val Tyr 135 140 145 tgc aaa ggc ccc tgt caa aga gtg cag ccg gga aaa
ctc agg gta cag 596 Cys Lys Gly Pro Cys Gln Arg Val Gln Pro Gly Lys
Leu Arg Val Gln 150 155 160 165 tgc agc acc tgc agg cag gca acg ctc
acc ttg acc cag gaa ttt ttc 644 Cys Ser Thr Cys Arg Gln Ala Thr Leu
Thr Leu Thr Gln Glu Phe Phe 170 175 180 ttt aaa tgt gga gca cac ccc
acc tct gac aag gaa aca cca gta gct 692 Phe Lys Cys Gly Ala His Pro
Thr Ser Asp Lys Glu Thr Pro Val Ala 185 190 195 ttg cac ctg atc gca
aca aat agt cgg aac atc act tgc att acg tgc 740 Leu His Leu Ile Ala
Thr Asn Ser Arg Asn Ile Thr Cys Ile Thr Cys 200 205 210 aca gac gtc
agg agc ccc gtc ctg gtt ttc cag tgc aac tcc cgc cac 788 Thr Asp Val
Arg Ser Pro Val Leu Val Phe Gln Cys Asn Ser Arg His 215 220 225 gtg
att tgc tta gac tgt ttc cac tta tac tgt gtg aca aga ctc aat 836 Val
Ile Cys Leu Asp Cys Phe His Leu Tyr Cys Val Thr Arg Leu Asn 230 235
240 245 gat cgg cag ttt gtt cac gac cct caa ctt ggc tac tcc ctg cct
tgt 884 Asp Arg Gln Phe Val His Asp Pro Gln Leu Gly Tyr Ser Leu Pro
Cys 250 255 260 gtg gct ggc tgt ccc aac tcc ttg att aaa gag ctc cat
cac ttc agg 932 Val Ala Gly Cys Pro Asn Ser Leu Ile Lys Glu Leu His
His Phe Arg 265 270 275 att ctg gga gaa gag cag tac aac cgg tac cag
cag tat ggt gca gag 980 Ile Leu Gly Glu Glu Gln Tyr Asn Arg Tyr Gln
Gln Tyr Gly Ala Glu 280 285 290 gag tgt gtc ctg cag atg ggg ggc gtg
tta tgc ccc cgc cct ggc tgt 1028 Glu Cys Val Leu Gln Met Gly Gly
Val Leu Cys Pro Arg Pro Gly Cys 295 300 305 gga gcg ggg ctg ctg ccg
gag cct gac cag agg aaa gtc acc tgc gaa 1076 Gly Ala Gly Leu Leu
Pro Glu Pro Asp Gln Arg Lys Val Thr Cys Glu 310 315 320 325 ggg ggc
aat ggc ctg ggc tgt ggg ttt gcc ttc tgc cgg gaa tgt aaa 1124 Gly
Gly Asn Gly Leu Gly Cys Gly Phe Ala Phe Cys Arg Glu Cys Lys 330 335
340 gaa gcg tac cat gaa ggg gag tgc agt gcc gta ttt gaa gcc tca gga
1172 Glu Ala Tyr His Glu Gly Glu Cys Ser Ala Val Phe Glu Ala Ser
Gly 345 350 355 aca act act cag gcc tac aga gtc gat gaa aga gcc gcc
gag cag gct 1220 Thr Thr Thr Gln Ala Tyr Arg Val Asp Glu Arg Ala
Ala Glu Gln Ala 360 365 370 cgt tgg gaa gca gcc tcc aaa gaa acc atc
aag aaa acc acc aag ccc 1268 Arg Trp Glu Ala Ala Ser Lys Glu Thr
Ile Lys Lys Thr Thr Lys Pro 375 380 385 tgt ccc cgc tgc cat gta cca
gtg gaa aaa aat gga ggc tgc atg cac 1316 Cys Pro Arg Cys His Val
Pro Val Glu Lys Asn Gly Gly Cys Met His 390 395 400 405 atg aag tgt
ccg cag ccc cag tgc agg ctc gag tgg tgc tgg aac tgt 1364 Met Lys
Cys Pro Gln Pro Gln Cys Arg Leu Glu Trp Cys Trp Asn Cys 410 415 420
ggc tgc gag tgg aac cgc gtc tgc atg ggg gac cac tgg ttc gac gtg
1412 Gly Cys Glu Trp Asn Arg Val Cys Met Gly Asp His Trp Phe Asp
Val 425 430 435 tagccagggc ggccgggcgc cccatcgcca catcctgggg
gagcataccc agtgtctacc 1472 ttcattttct aattctcttt tcaaacacac
acacacacgc gcgcgcgcgc acacacactc 1532 ttcaagtttt tttcaaagtc
caactacagc caaattgcag aagaaactcc tggatccctt 1592 tcactatgtc
catgaaaaac agcagagtaa aattacagaa gaagctcctg aatccctttc 1652
agtttgtcca cacaagacag cagagccatc tgcgacacca ccaacaggcg ttctcagcct
1712 ccggatgaca caaataccag agcacagatt caagtgcaat ccatgtatct
gtatgggtca 1772 ttctcacctg aattcgagac aggcagaatc agtagctgga
gagagagttc tcacatttaa 1832 tatcctgcct tttaccttca gtaaacacca
tgaagatgcc attgacaagg tgtttctctg 1892 taaaatgaac tgcagtgggt
tctccaaact agattcatgg ctttaacagt aatgttctta 1952 tttaaatttt
cagaaagcat ctattcccaa agaaccccag gcaatagtca aaaacatttg 2012
tttatcctta agaattccat ctatataaat cgcattaatc gaaataccaa ctatgtgtaa
2072 atcaacttgt cacaaagtga gaaattatga aagttaattt gaatgttgaa
tgtttgaatt 2132 acagggaaga aatcaagtta atgtactttc attccctttc
atgatttgca actttagaaa 2192 gaaattgttt ttctgaaagt atcaccaaaa
aatctatagt ttgattctga gtattcattt 2252 tgcaacttgg agattttgct
aatacatttg gctccactgt aaatttaata gataaagtgc 2312 ctataaagga
aacacgttta gaaatgattt caaaatgata ttcaatctta acaaaagtga 2372
acattattaa atcagaatct ttaaagagga gcctttccag aactaccaaa atgaagacac
2432 gcccgactct ctccatcaga agggtttata cccctttggc acaccctctc
tgtccaatct 2492 gcaagtccca gggagctctg cataccaggg gttccccagg
agagaccttc tcttaggaca 2552 gtaaactcac tagaatattc cttatgttga
catggattgg atttcagttc aatcaaactt 2612 tcagcttttt tttcagccat
tcacaacaca atcaaaagat taacaacact gcatgcggca 2672 aaccgcatgc
tcttacccac actacgcaga agagaaagta caaccactat
cttttgttct 2732 acctgtattg tctgacttct caggaagatc gtgaacataa
ctgagggcat gagtctcact 2792 agcacatgga ggcccttttg gatttagaga
ctgtaaatta ttaaatcggc aacagggctt 2852 ctctttttag atgtagcact gaaa
2876 4 437 PRT Homo sapiens 4 Met Ile Val Phe Val Arg Phe Asn Ser
Ser His Gly Phe Pro Val Glu 1 5 10 15 Val Asp Ser Asp Thr Ser Ile
Phe Gln Leu Lys Glu Val Val Ala Lys 20 25 30 Arg Gln Gly Val Pro
Ala Asp Gln Leu Arg Val Ile Phe Ala Gly Lys 35 40 45 Glu Leu Arg
Asn Asp Trp Thr Val Gln Asn Cys Asp Leu Asp Gln Gln 50 55 60 Ser
Ile Val His Ile Val Gln Arg Pro Trp Arg Lys Gly Gln Glu Met 65 70
75 80 Asn Ala Thr Gly Gly Asp Asp Pro Arg Asn Ala Ala Gly Gly Cys
Glu 85 90 95 Arg Glu Pro Gln Ser Leu Thr Arg Val Asp Leu Ser Ser
Ser Val Leu 100 105 110 Pro Gly Asp Ser Val Gly Leu Ala Val Ile Leu
His Thr Asp Ser Arg 115 120 125 Lys Asp Ser Pro Pro Ala Gly Ser Pro
Ala Gly Arg Ser Ile Tyr Asn 130 135 140 Ser Phe Tyr Val Tyr Cys Lys
Gly Pro Cys Gln Arg Val Gln Pro Gly 145 150 155 160 Lys Leu Arg Val
Gln Cys Ser Thr Cys Arg Gln Ala Thr Leu Thr Leu 165 170 175 Thr Gln
Glu Phe Phe Phe Lys Cys Gly Ala His Pro Thr Ser Asp Lys 180 185 190
Glu Thr Pro Val Ala Leu His Leu Ile Ala Thr Asn Ser Arg Asn Ile 195
200 205 Thr Cys Ile Thr Cys Thr Asp Val Arg Ser Pro Val Leu Val Phe
Gln 210 215 220 Cys Asn Ser Arg His Val Ile Cys Leu Asp Cys Phe His
Leu Tyr Cys 225 230 235 240 Val Thr Arg Leu Asn Asp Arg Gln Phe Val
His Asp Pro Gln Leu Gly 245 250 255 Tyr Ser Leu Pro Cys Val Ala Gly
Cys Pro Asn Ser Leu Ile Lys Glu 260 265 270 Leu His His Phe Arg Ile
Leu Gly Glu Glu Gln Tyr Asn Arg Tyr Gln 275 280 285 Gln Tyr Gly Ala
Glu Glu Cys Val Leu Gln Met Gly Gly Val Leu Cys 290 295 300 Pro Arg
Pro Gly Cys Gly Ala Gly Leu Leu Pro Glu Pro Asp Gln Arg 305 310 315
320 Lys Val Thr Cys Glu Gly Gly Asn Gly Leu Gly Cys Gly Phe Ala Phe
325 330 335 Cys Arg Glu Cys Lys Glu Ala Tyr His Glu Gly Glu Cys Ser
Ala Val 340 345 350 Phe Glu Ala Ser Gly Thr Thr Thr Gln Ala Tyr Arg
Val Asp Glu Arg 355 360 365 Ala Ala Glu Gln Ala Arg Trp Glu Ala Ala
Ser Lys Glu Thr Ile Lys 370 375 380 Lys Thr Thr Lys Pro Cys Pro Arg
Cys His Val Pro Val Glu Lys Asn 385 390 395 400 Gly Gly Cys Met His
Met Lys Cys Pro Gln Pro Gln Cys Arg Leu Glu 405 410 415 Trp Cys Trp
Asn Cys Gly Cys Glu Trp Asn Arg Val Cys Met Gly Asp 420 425 430 His
Trp Phe Asp Val 435 5 21 DNA Artificial sequence oligonucleotide
primer 5 agcctggtta agtccaagct g 21 6 22 DNA Artificial sequence
oligonucleotide primer 6 gaaggtccca tttttcgttt tc 22 7 15 PRT
Artificial sequence amino acid residue 7 Cys Xaa Cys Xaa Cys Xaa
His Xaa Cys Xaa Cys Xaa Cys Xaa Cys 1 5 10 15 8 15 PRT Artificial
sequence amino acid residue 8 Cys Xaa Cys Xaa Cys Xaa His Xaa Cys
Xaa Cys Xaa Cys Xaa Cys 1 5 10 15 9 20 DNA Homo sapiens
misc_feature (1)..(10) Exon 1 9 accatgatag gtacgtgggt 20 10 20 DNA
Homo sapiens Intron (1)..(10) Intron 10 ccttggtcag tgtttgtcag 20 11
20 DNA Homo sapiens misc_feature (1)..(10) Exon 2 11 gactgtgcag
gtgagtctcc 20 12 20 DNA Homo sapiens Intron (1)..(10) Intron 12
tcccaaacag aattgtgacc 20 13 20 DNA Homo sapiens misc_feature
(1)..(10) Exon 3 13 ggaagtccag gtaattggaa 20 14 20 DNA Homo sapiens
Intron (1)..(10) Intron 14 tcttctccag caggtagatc 20 15 20 DNA Homo
sapiens misc_feature (1)..(10) Exon 4 15 cttgacccag gtaaggaaat 20
16 20 DNA Homo sapiens Intron (1)..(10) Intron 16 tttcccaaag
ggtccatctt 20 17 20 DNA Homo sapiens misc_feature (1)..(10) Exon 5
17 gactagtgca gtaagtacct 20 18 20 DNA Homo sapiens Intron (1)..(10)
Intron 18 tttctttcag gaatttttct 20 19 20 DNA Homo sapiens
misc_feature (1)..(10) Exon 6 19 cagacgtcag gtaaggatct 20 20 20 DNA
Homo sapiens Intron (1)..(10) Intron 20 ctctctgcag gagccccgtc 20 21
20 DNA Homo sapiens misc_feature (1)..(10) Exon 7 21 ccttgtgtgg
gtaagtctag 20 22 20 DNA Homo sapiens Intron (1)..(10) Intron 22
tttccaacag ctggctgtcc 20 23 20 DNA Homo sapiens misc_feature
(1)..(10) Exon 8 23 agaagagcag gtgagtgagc 20 24 20 DNA Homo sapiens
Intron (1)..(10) Intron 24 ggttttgcag tacaaccggt 20 25 20 DNA Homo
sapiens misc_feature (1)..(10) Exon 9 25 gggctgtggg gtgagtactg 20
26 20 DNA Homo sapiens Intron (1)..(10) Intron 26 tcttttgcag
tttgccttct 20 27 20 DNA Homo sapiens misc_feature (1)..(10) Exon 10
27 aactactcag gtacagaatg 20 28 20 DNA Homo sapiens Intron (1)..(10)
Intron 28 gtttccccag gcctacagag 20 29 20 DNA Homo sapiens
misc_feature (1)..(10) Exon 11 29 gaaaaaaatg gtgagtctgt 20 30 20
DNA Homo sapiens Intron (1)..(10) Intron 30 cccccaacag gaggctgcat
20 31 23 DNA Artificial sequence oligonucleotide primer 31
tgatagtcat aactgtgtgt aag 23 32 23 DNA Artificial sequence
oligonucleotide primer 32 acagggaaca taaactctga tcc 23 33 22 DNA
Artificial sequence oligonucleotide primer 33 caacacacca ggcaccttca
ga 22 34 19 DNA Artificial sequence oligonucleotide primer 34
gtttgggaat gcgtgtttt 19 35 24 DNA Artificial sequence
oligonucleotide primer 35 agaattagaa aatgaaggta gaca 24 36 24 DNA
Artificial sequence oligonucleotide primer 36 gcgcggctgg cgccgctgcg
cgca 24 37 20 DNA Artificial sequence oligonucleotide primer 37
gcggcgcaga gaggctgtac 20 38 23 DNA Artificial sequence
oligonucleotide primer 38 atgttgctat caccatttaa ggg 23 39 23 DNA
Artificial sequence oligonucleotide primer 39 agattggcag cgcaggcggc
atg 23 40 21 DNA Artificial sequence oligonucleotide primer 40
acatgtcact tttgcttccc t 21 41 23 DNA Artificial sequence
oligonucleotide primer 41 aggccatgct ccatgcagac tgc 23 42 24 DNA
Artificial sequence oligonucleotide primer 42 acaagctttt aaagagtttc
ttgt 24 43 20 DNA Artificial sequence oligonucleotide primer 43
aggcaatgtg ttagtacaca 20 44 22 DNA Artificial sequence
oligonucleotide primer 44 acatgtctta aggagtacat tt 22 45 24 DNA
Artificial sequence oligonucleotide primer 45 tctctaattt cctggcaaac
agtg 24 46 23 DNA Artificial sequence oligonucleotide primer 46
agagattgtt tactgtggaa aca 23 47 23 DNA Artificial sequence
oligonucleotide primer 47 gagtgatgct atttttagat cct 23 48 24 DNA
Artificial sequence oligonucleotide primer 48 tgcctttcca cactgacagg
tact 24 49 24 DNA Artificial sequence oligonucleotide primer 49
tctgttcttc attagcatta gaga 24 50 23 DNA Artificial sequence
oligonucleotide primer 50 tgatagtcat aactgtgtgt aag 23 51 23 DNA
Artificial sequence oligonucleotide primer 51 actgtctcat tagcgtctat
ctt 23 52 20 DNA Artificial sequence oligonucleotide primer 52
gggtgaaatt tgcagtcagt 20 53 23 DNA Artificial sequence
oligonucleotide primer 53 aatataatcc cagcccatgt gca 23 54 22 DNA
Artificial sequence oligonucleotide primer 54 attgccaaat gcaacctmtg
tc 22 55 22 DNA Artificial sequence oligonucleotide primer 55
ttggaggaat gagtagggca tt 22 56 23 DNA Artificial sequence
oligonucleotide primer 56 acagggaaca taaactctga tcc 23 57 22 DNA
Artificial sequence oligonucleotide primer 57 caacacacca ggcaccttca
ga 22 58 19 DNA Artificial sequence oligonucleotide primer 58
gtttgggaat gcgtgtttt 19 59 24 DNA Artificial sequence
oligonucleotide primer 59 agaattagaa aatgaaggta gaca 24 60 76 PRT
Homo sapiens 60 Met Gln Ile Phe Val Lys Thr Leu Thr Gly Lys Thr Ile
Thr Leu Glu 1 5 10 15 Val Glu Pro Ser Asp Thr Ile Glu Asn Val Lys
Ala Lys Ile Gln Asp 20 25 30 Lys Glu Gly Ile Pro Pro Asp Gln Gln
Arg Leu Ile Phe Ala Gly Lys 35 40 45 Gln Leu Glu Asp Gly Arg Thr
Leu Ser Asp Tyr Asn Ile Gln Lys Glu 50 55 60 Ser Thr Leu His Leu
Val Leu Arg Leu Arg Gly Gly 65 70 75 61 76 PRT Saccharomyces sp. 61
Met Gln Ile Phe Val Lys Thr Leu Thr Gly Lys Thr Ile Thr Leu Glu 1 5
10 15 Val Glu Ser Ser Asp Thr Ile Asp Asn Val Lys Ser Lys Ile Gln
Asp 20 25 30 Lys Glu Gly Ile Pro Pro Asp Gln Gln Arg Leu Ile Phe
Ala Gly Lys 35 40 45 Gln Leu Glu Asp Gly Arg Thr Leu Ser Asp Tyr
Asn Ile Gln Lys Glu 50 55 60 Ser Thr Leu His Leu Val Leu Arg Leu
Arg Gly Gly 65 70 75 62 76 PRT Glycine sp. 62 Met Gln Ile Phe Val
Lys Thr Leu Thr Gly Lys Thr Ile Thr Leu Glu 1 5 10 15 Val Glu Ser
Ser Asp Thr Ile Asp Asn Val Lys Ala Lys Ile Gln Asp 20 25 30 Lys
Glu Gly Ile Pro Pro Asp Gln Gln Arg Leu Ile Phe Ala Gly Lys 35 40
45 Gln Leu Glu Asp Gly Arg Thr Leu Ala Asp Tyr Asn Ile Gln Lys Glu
50 55 60 Ser Thr Leu His Leu Val Leu Arg Leu Arg Gly Gly 65 70 75
63 33 DNA Homo sapiens 63 caccagctgg aagtccaggg tccatcttgc tgg 33
64 40 DNA Homo sapiens 64 tcccaaaggg tccatcttgc tgggatgatg
ttttaattcc 40 65 40 DNA Homo sapiens 65 tcccaaaggt ccatcttgct
gggatgatgt tttaattcca 40 66 38 DNA Homo sapiens Intron (1)..(8)
Intron 66 tcccaaag ggt cca tct tgc tgg gat gat gtt tta att 38 Gly
Pro Ser Cys Trp Asp Asp Val Leu Ile 1 5 10 67 10 PRT Homo sapiens
67 Gly Pro Ser Cys Trp Asp Asp Val Leu Ile 1 5 10 68 37 DNA Homo
sapiens Intron (1)..(8) Intron 68 tcccaaag gtc cat ctt gct ggg atg
atg ttt taatt 37 Val His Leu Ala Gly Met Met Phe 1 5 69 8 PRT Homo
sapiens 69 Val His Leu Ala Gly Met Met Phe 1 5 70 21 DNA Homo
sapiens 70 gggacagcca gccacacaag g 21
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