U.S. patent application number 15/601804 was filed with the patent office on 2017-11-09 for detection and treatment of polycystic kidney disease.
The applicant listed for this patent is The Johns Hopkins University School of Medicine. Invention is credited to Gregory G. Germino, Bunyong Phakdeekitcharoen, Terry J. Watnick.
Application Number | 20170321278 15/601804 |
Document ID | / |
Family ID | 26912733 |
Filed Date | 2017-11-09 |
United States Patent
Application |
20170321278 |
Kind Code |
A1 |
Germino; Gregory G. ; et
al. |
November 9, 2017 |
DETECTION AND TREATMENT OF POLYCYSTIC KIDNEY DISEASE
Abstract
Compositions useful for examining the PKD1 gene are provided. In
addition, methods for detecting mutations of the PKD1 gene, which
can be associated with autosomal dominant polycystic kidney disease
in humans, are provided. Methods for diagnosing a mutant PKD1 gene
sequence in a subject also are provided, as are methods of treating
a subject having a PKD1-associated disorder.
Inventors: |
Germino; Gregory G.; (Chevy
Chase, MD) ; Watnick; Terry J.; (Chevy Chase, MD)
; Phakdeekitcharoen; Bunyong; (Bangkok, TH) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Johns Hopkins University School of Medicine |
Baltimore |
MD |
US |
|
|
Family ID: |
26912733 |
Appl. No.: |
15/601804 |
Filed: |
May 22, 2017 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
13965083 |
Aug 12, 2013 |
9670543 |
|
|
15601804 |
|
|
|
|
11485062 |
Jul 11, 2006 |
8530161 |
|
|
13965083 |
|
|
|
|
09904968 |
Jul 13, 2001 |
7553644 |
|
|
11485062 |
|
|
|
|
60283691 |
Apr 13, 2001 |
|
|
|
60218261 |
Jul 13, 2000 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 13/00 20180101;
C12Q 1/6883 20130101; C12Q 2600/156 20130101 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] This invention was made with government support under Grant
Nos. DK48006, TW05393 and DK02562 awarded by the National
Institutes of Health. The government has certain rights in the
invention.
Claims
1. A method of selectively amplifying a region of the PKD1 gene,
but not the corresponding region of a PKD1 homolog, wherein said
PKD1 gene comprises SEQ ID NO:1, the method comprising amplifying a
DNA sample which contains the PKD1 gene with a set of eight
PKD1-specific primer pairs to obtain PKD1-specific amplification
products of the PKD1 gene, wherein the amplification products do
not contain PKD1 homolog sequence.
2. The method of claim 1, wherein said set of eight primer pairs
selectively hybridize to SEQ ID NO:1 and amplify portions of SEQ ID
NO:1 comprising about nucleotides 2043 to 4290; nucleotides 17907
to 22489; nucleotides 22218 to 26363; nucleotides 26246 to 30615;
nucleotides 30606 to 33957; nucleotides 36819 to 37140; nucleotides
37329 to 41258 and nucleotides 41508 to 47320.
3. The method of claim 2, wherein said set of eight primer pairs
comprise SEQ ID NOS:3 and 4; SEQ ID NOS:5 and 6; SEQ ID NOS:7 and
8; SEQ ID NOS:9 and 10; SEQ ID NOS:11 and 12; SEQ ID NOS:13 and 14;
SEQ ID NOS:15 and 16; and SEQ ID NOS:17 and 18.
4. The method of claim 1, wherein said PKD1-specific amplification
products encompass all of the exons within the PKD1 gene.
5. The method of claim 1, further comprising performing a nested
PCR reaction comprising contacting the PKD1-specific amplification
products of claim 79 with a second set of primers under conditions
suitable for nested PCR to obtain nested PCR products of the
PKD1-specific amplification products.
6. The method of claim 5, wherein the second primer pair is
selected from the group consisting of SEQ ID NOS:19 and 20; SEQ ID
NOS:21 and 22; SEQ ID NOS:23 and 24; SEQ ID NOS:25 and 26; SEQ ID
NOS:27 and 28; SEQ ID NOS:29 and 30; SEQ ID NOS:31 and 32; SEQ ID
NOS:33 and 34; SEQ ID NOS:35 and 36; SEQ ID NOS:37 and 38; SEQ ID
NOS:39 and 40; SEQ ID NOS:41 AND 42; SEQ ID NOS:43 and 44; SEQ ID
NOS:45 and 46; SEQ ID NOS:47 and 48; SEQ ID NOS:49 and 50; SEQ ID
NOS:51 and 61; primer pairs formed using consecutive primers set
forth in Table 2 as SEQ ID NOS:62 to 96, 113, and 97 to 112; or any
combination of pairs thereof.
7. The method of claim 5, further comprising a dilution step, to
remove genomic contamination from the amplification products, prior
to the nested PCR reaction.
8. The method of claim 1, wherein the nucleotide sequences of the
PKD1-specific amplification products obtained from a patient sample
are compared to the nucleotide sequence of PKD1-specific
amplification products from a control sample to determine if there
are one or more differences between the PKD1-specific amplification
products of the patient sample and the control sample, wherein the
presence of one or more differences in the sequence of the
PKD1-specific amplification products in the patient sample compared
to the control sample PKD1 sequence is indicative of a mutation in
the patient's PKD1 gene.
9. The method of claim 8, wherein the one or more differences are
selected from the group consisting of: nucleotide 474, wherein
nucleotide 474 is a T; nucleotide 487, wherein nucleotide 487 is an
A; nucleotide 3110, wherein nucleotide 3110 is a C; a position
corresponding to nucleotide 3336, wherein nucleotide 3336 is
deleted; nucleotide 3707, wherein nucleotide 3707 is an A;
nucleotide 4168, wherein nucleotide 4168 is a T; nucleotide 4885,
wherein nucleotide 4885 is an A; nucleotide 5168, wherein
nucleotide 5168 is a T; nucleotide 6058, wherein nucleotide 6058 is
a T; nucleotide 6078, wherein nucleotide 6078 is an A; nucleotide
6089, wherein nucleotide 6089 is a T; nucleotide 6195, wherein
nucleotide 6195 is an A; nucleotide 6326, wherein nucleotide 6326
is a T; a position corresponding to nucleotides 7205-7211, wherein
nucleotides 7205 to 7211 are deleted; nucleotide 7376, wherein
nucleotide 7376 is a C; a nucleotide sequence corresponding to
nucleotides 7535 to 7536, wherein a GCG nucleotide sequence is
inserted between nucleotides 7535 and 7536; nucleotide 7415,
wherein nucleotide 7415 is a T; nucleotide 7433, wherein nucleotide
7433 is a T; nucleotide 7696, wherein nucleotide 7696 is a T;
nucleotide 7883, wherein nucleotide 7883 is a T; nucleotide 8021,
wherein nucleotide 8021 is an A; a nucleotide sequence
corresponding to nucleotide 8159 to 8160, wherein nucleotides
8159-8160 are deleted; nucleotide 8298, wherein nucleotide 8298 is
a G; nucleotide 9164, wherein nucleotide 9164 is a G; nucleotide
9213, wherein nucleotide 9213 is an A; nucleotide 9326, wherein
nucleotide 9326 is a T; nucleotide 9367, wherein nucleotide 9367 is
a T; nucleotide 10064, wherein nucleotide 10064 is an A; nucleotide
10143, wherein nucleotide 10143 is a G; nucleotide 10234, wherein
nucleotide 10234 is a C; nucleotide 10255, wherein nucleotide 10255
is a T; or a combination thereof.
10. The method of claim 5, wherein the nucleotide sequence of the
nested PCR products of the PKD1-specific amplification products
obtained from a patient sample are compared to the nucleotide
sequence of the nested PCR products of the PKD1-specific
amplification products from a control sample to determine if there
are one or more differences between the nucleotide sequence of the
nested PCR products of the patient sample and the control sample,
wherein the presence of one or more differences in the nucleotide
sequence of the nested PCR products in the patient sample compared
to the nucleotide sequence of the nested PCR products in the
control sample is indicative of a mutation in the patient's PKD1
gene.
11. The method of claim 10, wherein the one or more differences are
selected from the group consisting of: nucleotide 474, wherein
nucleotide 474 is a T; nucleotide 487, wherein nucleotide 487 is an
A; nucleotide 3110, wherein nucleotide 3110 is a C; a position
corresponding to nucleotide 3336, wherein nucleotide 3336 is
deleted; nucleotide 3707, wherein nucleotide 3707 is an A;
nucleotide 4168, wherein nucleotide 4168 is a T; nucleotide 4885,
wherein nucleotide 4885 is an A; nucleotide 5168, wherein
nucleotide 5168 is a T; nucleotide 6058, wherein nucleotide 6058 is
a T; nucleotide 6078, wherein nucleotide 6078 is an A; nucleotide
6089, wherein nucleotide 6089 is a T; nucleotide 6195, wherein
nucleotide 6195 is an A; nucleotide 6326, wherein nucleotide 6326
is a T; a position corresponding to nucleotides 7205-7211, wherein
nucleotides 7205 to 7211 are deleted; nucleotide 7376, wherein
nucleotide 7376 is a C; a nucleotide sequence corresponding to
nucleotides 7535 to 7536, wherein a GCG nucleotide sequence is
inserted between nucleotides 7535 and 7536; nucleotide 7415,
wherein nucleotide 7415 is a T; nucleotide 7433, wherein nucleotide
7433 is a T; nucleotide 7696, wherein nucleotide 7696 is a T;
nucleotide 7883, wherein nucleotide 7883 is a T; nucleotide 8021,
wherein nucleotide 8021 is an A; a nucleotide sequence
corresponding to nucleotide 8159 to 8160, wherein nucleotides
8159-8160 are deleted; nucleotide 8298, wherein nucleotide 8298 is
a G; nucleotide 9164, wherein nucleotide 9164 is a G; nucleotide
9213, wherein nucleotide 9213 is an A; nucleotide 9326, wherein
nucleotide 9326 is a T; nucleotide 9367, wherein nucleotide 9367 is
a T; nucleotide 10064, wherein nucleotide 10064 is an A; nucleotide
10143, wherein nucleotide 10143 is a G; nucleotide 10234, wherein
nucleotide 10234 is a C; nucleotide 10255, wherein nucleotide 10255
is a T; or a combination thereof.
12. The method of claim 6, wherein the sequences of the
PKD1-specific amplification products obtained from a patient sample
are compared to the sequence of PKD1-specific amplification
products from a control sample to determine if there are one or
more differences between the PKD1-specific amplification products
of the patient sample and the control sample, and wherein the
presence of one or more differences in the sequence of the
PKD1-specific amplification products compared to the control PKD1
sequence is indicative of a mutation in the PKD1 gene.
13. The method of claim 12, wherein the one or more differences are
selected from the group consisting of: nucleotide 474, wherein
nucleotide 474 is a T; nucleotide 487, wherein nucleotide 487 is an
A; nucleotide 3110, wherein nucleotide 3110 is a C; a position
corresponding to nucleotide 3336, wherein nucleotide 3336 is
deleted; nucleotide 3707, wherein nucleotide 3707 is an A;
nucleotide 4168, wherein nucleotide 4168 is a T; nucleotide 4885,
wherein nucleotide 4885 is an A; nucleotide 5168, wherein
nucleotide 5168 is a T; nucleotide 6058, wherein nucleotide 6058 is
a T; nucleotide 6078, wherein nucleotide 6078 is an A; nucleotide
6089, wherein nucleotide 6089 is a T; nucleotide 6195, wherein
nucleotide 6195 is an A; nucleotide 6326, wherein nucleotide 6326
is a T; a position corresponding to nucleotides 7205-7211, wherein
nucleotides 7205 to 7211 are deleted; nucleotide 7376, wherein
nucleotide 7376 is a C; a nucleotide sequence corresponding to
nucleotides 7535 to 7536, wherein a GCG nucleotide sequence is
inserted between nucleotides 7535 and 7536; nucleotide 7415,
wherein nucleotide 7415 is a T; nucleotide 7433, wherein nucleotide
7433 is a T; nucleotide 7696, wherein nucleotide 7696 is a T;
nucleotide 7883, wherein nucleotide 7883 is a T; nucleotide 8021,
wherein nucleotide 8021 is an A; a nucleotide sequence
corresponding to nucleotide 8159 to 8160, wherein nucleotides
8159-8160 are deleted; nucleotide 8298, wherein nucleotide 8298 is
a G; nucleotide 9164, wherein nucleotide 9164 is a G; nucleotide
9213, wherein nucleotide 9213 is an A; nucleotide 9326, wherein
nucleotide 9326 is a T; nucleotide 9367, wherein nucleotide 9367 is
a T; nucleotide 10064, wherein nucleotide 10064 is an A; nucleotide
10143, wherein nucleotide 10143 is a G; nucleotide 10234, wherein
nucleotide 10234 is a C; nucleotide 10255, wherein nucleotide 10255
is a T; or a combination thereof.
14. A method of detecting mutations in a PKD1 gene comprising SEQ
ID NO:1, comprising the steps of: amplifying genomic DNA contained
in test and control samples with a set of eight PKD1-specific
primer pairs to obtain PKD1-specific amplification products of the
PKD1 gene; diluting the PKD1-specific amplification products to
remove genomic contamination from the amplification products;
contacting the diluted amplification products with a second set of
primers under conditions suitable for nested PCR to obtain nested
PCR products of the PKD1-specific amplification products; and
comparing the nucleotide sequence of the nested PCR products in the
test sample with the nucleotide sequence of the nested PCR products
in the control sample, whereby the presence of one or more
differences in the nucleotide sequence of the nested PCR products
of the test sample compared to the nucleotide sequence of the
nested PCR products of the control sample is indicative of a
mutation in the PKD1 gene.
15. The method of claim 14, wherein the one or more mutations are
selected from the group consisting of: nucleotide 474, wherein
nucleotide 474 is a T; nucleotide 487, wherein nucleotide 487 is an
A; nucleotide 3110, wherein nucleotide 3110 is a C; a position
corresponding to nucleotide 3336, wherein nucleotide 3336 is
deleted; nucleotide 3707, wherein nucleotide 3707 is an A;
nucleotide 4168, wherein nucleotide 4168 is a T; nucleotide 4885,
wherein nucleotide 4885 is an A; nucleotide 5168, wherein
nucleotide 5168 is a T; nucleotide 6058, wherein nucleotide 6058 is
a T; nucleotide 6078, wherein nucleotide 6078 is an A; nucleotide
6089, wherein nucleotide 6089 is a T; nucleotide 6195, wherein
nucleotide 6195 is an A; nucleotide 6326, wherein nucleotide 6326
is a T; a position corresponding to nucleotides 7205-7211, wherein
nucleotides 7205 to 7211 are deleted; nucleotide 7376, wherein
nucleotide 7376 is a C; a nucleotide sequence corresponding to
nucleotides 7535 to 7536, wherein a GCG nucleotide sequence is
inserted between nucleotides 7535 and 7536; nucleotide 7415,
wherein nucleotide 7415 is a T; nucleotide 7433, wherein nucleotide
7433 is a T; nucleotide 7696, wherein nucleotide 7696 is a T;
nucleotide 7883, wherein nucleotide 7883 is a T; nucleotide 8021,
wherein nucleotide 8021 is an A; a nucleotide sequence
corresponding to nucleotide 8159 to 8160, wherein nucleotides
8159-8160 are deleted; nucleotide 8298, wherein nucleotide 8298 is
a G; nucleotide 9164, wherein nucleotide 9164 is a G; nucleotide
9213, wherein nucleotide 9213 is an A; nucleotide 9326, wherein
nucleotide 9326 is a T; nucleotide 9367, wherein nucleotide 9367 is
a T; nucleotide 10064, wherein nucleotide 10064 is an A; nucleotide
10143, wherein nucleotide 10143 is a G; nucleotide 10234, wherein
nucleotide 10234 is a C; nucleotide 10255, wherein nucleotide 10255
is a T; or a combination thereof.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation application of U.S.
application Ser. No. 13/965,083 filed Aug. 12, 2013, now issued as
U.S. Pat. No. 9,670,543; which is a continuation application of
U.S. application Ser. No. 11/485,062 filed Jul. 11, 2006, now
issued as U.S. Pat. No. 8,530,161; which is a continuation
application of U.S. application Ser. No. 09/904,968 filed Jul. 13,
2001, now issued as U.S. Pat. No. 7,553,644; which claims the
benefit under 35 USC .sctn.119(e) to U.S. Application Ser. No.
60/283,691 filed Apr. 13, 2001 and to U.S. Application Ser. No.
60/218,261 filed Jul. 13, 2000, both now expired. The disclosure of
each of the prior applications is considered part of and is
incorporated by reference in the disclosure of this
application.
BACKGROUND OF THE INVENTION
Field of the Invention
[0003] The present invention relates generally to the diagnosis and
treatment of polycystic kidney disease and more specifically to
probes and agents useful in diagnosing and treating polycystic
kidney disease and related disorders.
Background Information
[0004] Autosomal dominant polycystic kidney disease (ADPKD), also
called adult-onset polycystic kidney disease, is one of the most
common hereditary disorders in humans, affecting approximately one
individual in a thousand. The prevalence in the United States is
greater than 500,000, with 6,000 to 7,000 new cases detected yearly
(Striker et al., Am. J. Nephrol. 6:161-164, 1986; Iglesias et al.,
Am. J. Kid. Dis. 2:630-639, 1983). The disease is considered to be
a systemic disorder, characterized by cyst formation in the ductal
organs such as kidney, liver, and pancreas, as well as by
gastrointestinal, cardiovascular, and musculoskeletal
abnormalities, including colonic diverticulitis, berry aneurysms,
hernias, and mitral valve prolapse (Gabow et al., Adv. Nephrol.
18:19-32, 1989; Gabow, New Eng. J. Med. 329:332-342, 1993).
[0005] The most prevalent and obvious symptom of ADPKD is the
formation of kidney cysts, which result in grossly enlarged kidneys
and a decrease in renal-concentrating ability. In approximately
half of ADPKD patients, the disease progresses to end-stage renal
disease, and ADPKD is responsible for 4-8% of the renal dialysis
and transplantation cases in the United States and Europe (Proc.
Eur. Dialysis and Transplant Assn., Robinson and Hawkins, eds.,
17:20, 1981).
[0006] Few diagnostics are available for the identification and
characterization of mutations of the PKD1 gene, which is located on
human chromosome 16. A major factor contributing to the difficulty
in identifying and characterizing mutations of the PKD1 gene is
that greater than 70% of the length of the PKD1 gene is replicated
on chromosome 16 and elsewhere, resulting in at least six PKD1
homologs. Significantly, the PKD1 homologs share a very high
sequence identity with the PKD1 gene, including sequences having
greater than 95% identity with the PKD1 gene. As such,
oligonucleotides that have been examined for use as specific
probes, or as primers for amplification, of PKD1 gene sequences
have been found to cross-hybridize with the PKD1 homologs, and the
inability to identify PKD1 locus specific probes has prevented
accurate analysis of PKD1 gene mutations.
[0007] The identification and characterization of PKD1 gene
mutations have been further hindered, in part, because
transcription of the PKD1 gene results in production of a 14
kilobase (kb) mRNA, which is highly GC-rich. In addition, unlike
the remainder of the PKD1 gene, which is extremely compact
(approximately 13.5 kb mRNA coded within approximately 30 kb
genomic DNA), exon 1 is separated from the rest of the gene by an
intron of approximately 19 kb. Thus, previous investigators have
simply placed the 5' anchor primer within the first intron and used
it as a link to more 3' sequences. Exon 1 has several other
features that have been major obstacles to its amplification,
including an extremely high GC content (approximately 85%), and the
ability to replicate with high fidelity in PKD1 gene homologs.
Furthermore, no effective method for DNA based analysis of PKD1
gene exon 22, which is flanked on both ends by introns that contain
lengthy polypyrimidine tracts. Accordingly, very few positions
within the replicated segment and flanking exon 22 are suitable for
the design of PKD1-specific primers.
[0008] A few oligonucleotides useful for examining regions of the
human PKD1 gene, have been described. For example, the primer set
forth below as SEQ ID NO:11 has been described in U.S. Pat. No.
6,017,717, and the primer set forth as SEQ ID NO:18 has been
described by Watnick et al. (Hum. Mol. Genet. 6:1473-1481, 1997).
Also, the primers set forth below as SEQ ID NOS:9, 10, 49 to 51,
and 61 to 105 have been described by Watnick et al. (Am. J. Hum.
Genet. 65:1561-1571, 1999). The primers set forth below as SEQ ID
NOS: 9 and 10 and SEQ ID NOS: 11 and 12 also were more recently
described by Phakdeekitcharoen et al. (Kidney International
58:1400-1412, 2000). In addition, a primer set forth as SEQ ID
NO:13 in U.S. Pat. No. 6,071,717 has a nucleotide sequence that is
substantially identical to that set forth below as SEQ ID NO:10,
and a primer designated TWR2 by Watnick et al. (Mol. Cell
2:247-251, 1998) has a nucleotide sequence that is substantially
identical to that set forth below as SEQ ID NO:12.
[0009] Despite the large number of families having diseases
associated with PKD1 gene mutations, the potential clinical and
scientific impact of mutation studies, and the availability of a
genomic structure, the fact that only a relatively small number of
PKD1 mutations have been described demonstrates the relative
paucity of data due to the complicated genomic structure of the
PKD1 gene. Thus, there exists a need for diagnostic methods
suitable for examining the PKD1 gene and for identifying disorders
related to PKD1 gene mutations. The present invention satisfies
this need and provides additional advantages.
SUMMARY OF THE INVENTION
[0010] The present invention provides compositions and methods that
allow for the selective examination of the human PKD1 gene,
including the detection and identification of PKD1 gene mutations.
For example, the compositions of the invention include
oligonucleotide primers that are useful for selectively amplifying
a region of a PKD1 gene, but not a corresponding region of a PKD1
homolog. Accordingly, the present invention relates to a PKD1 gene
specific primer, which can be one of a primer pair. A primer of the
invention includes a 5' region and adjacent PKD1-specific 3'
region, wherein the 5' region has a nucleotide sequence that can
hybridize to a PKD1 gene sequence and, optionally, to a PKD1
homolog sequence, and the 3' region has a nucleotide sequence that
selectively hybridizes only to a PKD1 gene sequence, and
particularly not to a PKD1 gene homolog sequence, except that a
primer of the invention does not have a sequence as set forth in
SEQ ID NO:11, SEQ ID NO:18, SEQ ID NO:52, or SEQ ID NO:60. A 5'
region of a primer of the invention generally contains at least
about ten contiguous nucleotides, and the 3' region contains at
least one 3' terminal nucleotide, wherein the at least one 3'
terminal nucleotide is identical to a nucleotide that is 5' and
adjacent to the nucleotide sequence of the PKD1 gene to which the
5' region of the primer can hybridize, and is different from a
nucleotide that is 5' and adjacent to a nucleotide sequence of the
PKD1 homolog to which the 5' region of the primer can hybridize.
Generally, the primer includes a 5' region of about 14 to 18
nucleotides and a 3' region of about 2 to 6 nucleotides,
particularly about 2 to 4 nucleotides. For example, a primer of the
invention can have a sequence as set forth in any of SEQ ID NOS:3
to 10, 12 to 17, 19 to 51 and 61 to 113.
[0011] The present invention also relates to an isolated mutant
PKD1 polynucleotide, or an oligonucleotide portion thereof. The
polynucleotides of the invention are exemplified by mutation of SEQ
ID NO:1, which appear to be normal variants that are not associated
with a PKD1-associated disorder, for example, a polynucleotide or
oligonucleotide that includes nucleotide 474, wherein nucleotide
474 is a T; nucleotide 487, wherein nucleotide 487 is an A;
nucleotide 9367, wherein nucleotide 9367 is a T; nucleotide 10143,
wherein nucleotide 10143 is a G; nucleotide 10234, wherein
nucleotide 10234 is a C; nucleotide 10255, wherein nucleotide 10255
is a T; or a combination thereof; and by mutations of SEQ ID NO:1
that are associated with a PKD1-associated disorder, for example, a
polynucleotide or oligonucleotide that includes nucleotide 3110 of
SEQ ID NO:1, wherein nucleotide 3110 is a C; nucleotide 8298 of SEQ
ID NO:1, wherein nucleotide 8298 is a G; nucleotide 9164 of SEQ ID
NO:1, wherein nucleotide 9164 is a G; nucleotide 9213 of SEQ ID
NO:1, wherein nucleotide 9213 is an A; nucleotide 9326 of SEQ ID
NO:1, wherein nucleotide 9326 is a T; nucleotide 10064 of SEQ ID
NO:1, wherein nucleotide 10064 is an A; or a combination thereof.
The invention also provides a vector containing such a
polynucleotide, or an oligonucleotide portion thereof, and provides
a host cell containing such a polynucleotide or oligonucleotide, or
vector.
[0012] A PKD1-specific primer of the invention is exemplified by an
oligonucleotide that can selectively hybridize to a nucleotide
sequence that flanks and is within about fifty nucleotides of a
nucleotide sequence selected from about nucleotides 2043 to 4209;
nucleotides 17907 to 22489; nucleotides 22218 to 26363; nucleotides
26246 to 30615; nucleotides 30606 to 33957; nucleotides 36819 to
37140; nucleotides 37329 to 41258; and nucleotides 41508 to 47320
of SEQ ID NO:1. The primer, which can be one of a primer pair, can
have a nucleotide sequence substantially identical to any of SEQ ID
NOS: 3 to 18, provided that when the primer is not one of a primer
pair, the primer does not have a sequence as set forth in SEQ ID
NO:11, SEQ ID NO:18, SEQ ID NO:52, or SEQ ID NO:60. Accordingly,
the present invention further relates to a primer pair that can
amplify a portion of a PKD1 gene, for example, the wild type PKD1
gene set forth as SEQ ID NO:1, wherein the amplification product
can include about nucleotides 2043 to 4209; nucleotides 17907 to
22489; nucleotides 22218 to 26363; nucleotides 26246 to 30615;
nucleotides 30606 to 33957; nucleotides 36819 to 37140; nucleotides
37329 to 41258; nucleotides 41508 to 47320; or a combination
thereof. A primer pair of the invention is useful for performing
PKD1-specific amplification of a portion of a PKD1 gene.
[0013] Primer pairs of the invention are exemplified by a pair
including at least one forward primer and at least one reverse
primer of the oligonucleotides sequences set forth in SEQ ID NOS:3
to 18 or a sequence substantially identical thereto. In one
embodiment, the primer pair includes SEQ ID NOS:3 and 4; SEQ ID
NOS:5 and 6; SEQ ID NOS:7 and 8; SEQ ID NOS:9 and 10; SEQ ID NOS:11
and 12; SEQ ID NOS:13 and 14; SEQ ID NOS:15 and 16; SEQ ID NOS:17
and 18; or SEQ ID NOS:9 and 113. Also provided are primer pairs
useful for performing nested amplification of a PKD1-specific
amplification product of a PKD1 gene, for example, the primer pairs
set forth as SEQ ID NOS:19 and 20; SEQ ID NOS:21 and 22; SEQ ID
NOS:23 and 24; SEQ ID NOS:25 and 26; SEQ ID NOS:27 and 28; SEQ ID
NOS:29 and 30; SEQ ID NOS:31 and 32; SEQ ID NOS:33 and 34; SEQ ID
NOS:35 and 36; SEQ ID NOS:37 and 38; SEQ ID NOS:39 and 40; SEQ ID
NOS:41 and 42; SEQ ID NOS:43 and 44; SEQ ID NOS:45 and 46; SEQ ID
NOS:47 and 48; SEQ ID NOS:49 and 50; SEQ ID NOS: 51 and 61; SEQ ID
NOS:62 and 63; SEQ ID NOS:64 and 65; SEQ ID NOS:66 and 67; SEQ ID
NOS:68 and 69; SEQ ID NOS:70 and 71; SEQ ID NOS:72 and 73; SEQ ID
NOS:74 and 75; SEQ ID NOS:76 and 77; SEQ ID NOS:78 and 79; SEQ ID
NOS:80 and 81; SEQ ID NOS:82 and 83; SEQ ID NOS:84 and 85; SEQ ID
NOS:86 and 87; SEQ ID NOS:88 and 89; SEQ ID NOS:90 and 91; SEQ ID
NOS:92 and 93; SEQ ID NOS:94 and 95; SEQ ID NOS:96 and 113; SEQ ID
NOS:97 and 98; SEQ ID NOS:99 and 100; SEQ ID NOS:101 and 102; SEQ
ID NOS:103 and 104; SEQ ID NOS: 105 and 106; SEQ ID NOS:107 and
108; SEQ ID NOS:109 and 110; or SEQ ID NOS:111 and 112. In another
embodiment, the invention relates to a plurality of primer pairs,
which can include two or more primer pairs that are useful for
generating two or more PKD1-specific amplification products of a
PKD1 gene; or can include two or more primer pairs that are useful
for generating a PKD1-specific amplification product of a PKD1 gene
and for generating a nested amplification product of the
PKD1-specific amplification product.
[0014] The present invention also relates to a purified mutant PKD1
polypeptide, or a peptide portion thereof, comprising an amino acid
sequence of a mutant of SEQ ID NO:2. A mutant PKD1 polypeptide, or
peptide portion thereof can be substantially identical to a
sequence of SEQ ID NO:2 and, for example, include amino acid
residue 88 of SEQ ID NO:2, wherein residue 88 is a V; residue 967
of SEQ ID NO:2, wherein residue 967 is an R; residue 2696 of SEQ ID
NO:2, wherein residue 2696 is an R; residue 2985 of SEQ ID NO:2,
wherein residue 2985 is a G; residue 3039 of SEQ ID NO:2, wherein
residue 3039 is a C; residue 3285 of SEQ ID NO:2, wherein residue
3285 is an I; or residue 3311 of SEQ ID NO:2, wherein residue 3311
is an R; or can include residue 3000 of a truncated mutant PKD1
polypeptide ending at amino acid residue 3000 with respect to SEQ
ID NO:2, wherein residue 3001 is absent (and the mutant PKD1
polypeptide is truncated) due to the presence of a STOP codon in
the encoding mutant PKD1 polynucleotide; or a combination of such
mutations. Also provided is a purified antibody that specifically
binds to a mutant PKD1 polypeptide, or to a peptide thereof.
[0015] The present invention further relates to a primer or an
oligonucleotide of the invention immobilized to a solid support. In
addition, the primer or oligonucleotide can be one of a plurality
of primers, oligonucleotides, or a combination thereof, each of
which is immobilized to a solid support. The solid support can be
any support, including, for example, a microchip, in which case,
the primers, oligonucleotides, or combination thereof can be
arranged in array, particularly an addressable array. The primers,
oligonucleotides, or combination thereof also can be degenerate
with respect to each other, and specific for a wild type PKD1
polynucleotide, a mutant PKD1 polynucleotide, including a variant,
or combinations thereof, and, therefore, provide a means for
multiplex analysis. Accordingly, the present invention provides
compositions comprising one or a plurality of immobilized primers
or oligonucleotides of the invention, or combinations thereof.
[0016] The present invention also relates to a method of detecting
a PKD1 polynucleotide in a sample, wherein the PKD1 polynucleotide
is a wild type PKD1 polynucleotide having a sequence as set forth
in SEQ ID NO:1, or a mutant PKD1 polynucleotide, which can be a
variant PKD1 polynucleotide that has a sequence different from SEQ
ID NO:1 but is not associated with a PKD1-associated disorder or
can be a mutant PKD1 polynucleotide that is associated with a
PKD1-associated disorder. A method of the invention can be
performed, for example, by contacting nucleic acid molecules in a
sample suspected of containing a PKD1 polynucleotide with at least
one primer pair under conditions suitable for amplification of a
PKD1 polynucleotide by the primer pair; and generating a
PKD1-specific amplification product under said conditions, thereby
detecting a PKD1 polynucleotide in the sample. The primer pair can
be any primer pair as disclosed herein, for example, a primer pair
such as SEQ ID NOS:3 and 4; SEQ ID NOS:5 and 6; SEQ ID NOS:7 and 8;
SEQ ID NOS:9 and 10; SEQ ID NOS:11 and 12; SEQ ID NOS:13 and 14;
SEQ ID NOS:15 and 16; SEQ ID NOS:17 and 18; or SEQ ID NOS:9 and
113; or can be a combination of such primer pairs.
[0017] A method of detecting a PKD1 polynucleotide can further
include, upon generating a PKD1-specific amplification product,
contacting the amplification product with at least a second primer
pair, under conditions suitable for nested amplification of the
PKD1-specific amplification product by the second primer pair, and
generating a nested amplification product. The second primer pair
can be any primer pair that can produce a nested amplification
product of the PKD1-specific amplification product, for example, a
second primer pair such as SEQ ID NOS:19 and 20; SEQ ID NOS:21 and
22; SEQ ID NOS:23 and 24; SEQ ID NOS:25 and 26; SEQ ID NOS:27 and
28; SEQ ID NOS:29 and 30; SEQ ID NOS:31 and 32; SEQ ID NOS:33 and
34; SEQ ID NOS:35 and 36; SEQ ID NOS:37 and 38; SEQ ID NOS:39 and
40; SEQ ID NOS:41 and 42; SEQ ID NOS:43 and 44; SEQ ID NOS:45 and
46; SEQ ID NOS:47 and 48; SEQ ID NOS:49 and 50; SEQ ID NOS:51 and
61; primer pairs formed using consecutive primers set forth in
Table 2 as SEQ ID NOS:62 to 96, 113, and 97 to 112; or a
combination thereof.
[0018] Upon detecting a PKD1 polynucleotide in a sample according
to a method of the invention, an additional step of detecting the
presence or absence of a mutation in an amplification product of
the PKD1 polynucleotide in the sample as compared to a
corresponding nucleotide sequence in SEQ ID NO:1. As such, a method
of the invention provides a means to identify a PKD1 polynucleotide
in a sample as a mutant PKD1 polynucleotide or a wild type PKD1
polynucleotide, wherein detecting the absence of a mutation in the
amplification product identifies the PKD1 polynucleotide in the
sample as a wild type PKD1 polynucleotide, and wherein detecting
the presence of a mutation in the amplification product identifies
the PKD1 polynucleotide in the sample as a mutant PKD1
polynucleotide, which can be a variant PKD1 polynucleotide, or can
be mutant PKD1 polynucleotide associated with a PKD1-associated
disorder, the latter of which are exemplified by a polynucleotide
that is substantially identical to SEQ ID NO:1, and wherein at
least nucleotide 474 is a T; nucleotide 487 is an A; nucleotide
3110 is a C; nucleotide 8298 is a G; nucleotide 9164 is a G;
nucleotide 9213 is an A; nucleotide 9326 is a T; nucleotide 9367 is
a T; nucleotide 10064 is an A; nucleotide 10143 is a G; nucleotide
10234 is a C; or nucleotide 10255 is a T.
[0019] The presence or absence of a mutation in an amplification
product generated according to a method of the invention can be
detected any method useful for detecting a mutation. For example,
the nucleotide sequence of the amplification product can be
determined, and can be compared to the corresponding nucleotide
sequence of SEQ ID NO:1. The melting temperature of the
amplification product also can be determined, and can be compared
to the melting temperature of a corresponding double stranded
nucleotide sequence of SEQ ID NO:1. The melting temperature can be
determined using a method such as denaturing high performance
liquid chromatography.
[0020] An advantage of a method of the invention is that a large
number of samples can be examined serially or in parallel.
Accordingly, a method of the invention can be performed with
respect to a plurality of samples, and can be performed using a
high throughput format, for example, by organizing the samples of a
plurality of samples in an array such as in an array is on a
microchip. The method can further include detecting the presence or
absence of a mutation in an amplification product of the samples of
the plurality of samples, for example, by determining the melting
temperature of the amplification product and comparing it to the
melting temperature of a corresponding nucleotide sequence of SEQ
ID NO:1 using a method such as denaturing high performance liquid
chromatography, or the presence or absence of a mutation can be
performed using any method useful for such a purpose, for example,
matrix-assisted laser desorption time of flight mass spectrometry
or high throughput conformation-sensitive gel electrophoresis, each
of which is readily adaptable to a high throughput analysis
format.
[0021] In another embodiment, the presence or absence of a mutation
in an amplification product can be detected by contacting the
amplification product with the oligonucleotide of the invention,
under condition suitable for selective hybridization of the
oligonucleotide to an identical nucleotide sequence; and detecting
the presence or absence of selective hybridization of the
oligonucleotide to the amplification product. Using such a method
detecting the presence of selective hybridization identifies the
PKD1 polynucleotide in the sample as a mutant PKD1 polynucleotide,
and detecting the absence of selective hybridization identifies the
PKD1 polynucleotide as a wild type PKD1 polynucleotide. Where an
absence of a mutation is detected, the PKD1 polynucleotide in the
sample is identified as a wild type PKD1 polynucleotide. In
comparison, where the presence of a mutation is identified, the
mutant PKD1 polynucleotide so identified can be further examined to
determine whether the mutant PKD1 polynucleotide is a variant PKD1
polynucleotide, which is associated with a normal phenotype with
respect to PKD1, for example, where the amplification product has a
nucleotide sequence substantially identical to SEQ ID NO:1, and
including C474T, G487A, G4885A; C6058T; G6195A; T7376C; C7696T;
G8021A; C9367T, A10143G, T10234C, or a combination thereof, or is a
mutant PKD1 polynucleotide associated with a PKD1-associated
disorder, for example, where the amplification product has a
nucleotide sequence substantially identical to SEQ ID NO:1, and
including T3110C, G3707A; T6078A; C7433T; T8298G; A9164G; G9213A,
C9326T; G10064A; an insertion of GCG between nucleotides G7535 and
A7536; or a combination thereof, each of which is associated with
ADPKD (see Example 2; see, also, Phakdeekitcharoen et al., Kidney
International 58:1400-1412, 2000, which is incorporated herein by
reference).
[0022] The present invention further relates to a method of
detecting the presence of a mutant PKD1 polynucleotide in a sample.
In one embodiment, a method of the invention is performed by
amplifying a nucleic acid sequence in a sample suspected of
containing a mutant PKD1 polynucleotide using a primer pair of the
invention, for example, a primer pair selected from SEQ ID NOS:3
and 4; SEQ ID NOS:5 and 6; SEQ ID NOS:7 and 8; SEQ ID NOS:9 and 10;
SEQ ID NOS:11 and 12; SEQ ID NOS:13 and 14; SEQ ID NOS:15 and 16;
SEQ ID NOS:17 and 18; or SEQ ID NOS:9 and 113, thereby obtaining a
PKD1-specific amplification product of a PKD1 gene sequence; and
detecting a mutant PKD1 polynucleotide in the amplification
product. The mutant PKD1 nucleotide in the amplification product
can be detected using any method useful for detecting a mutation in
a polynucleotide, for example, using denaturing high performance
liquid chromatograph. In another embodiment, a method of the
invention is performed by contacting a sample suspected of
containing a mutant PKD1 polynucleotide with a probe comprising an
isolated polynucleotide of the invention, or an oligonucleotide
portion thereof, under conditions such that the probe selectively
hybridizes to a mutant PKD1 polynucleotide, and detecting specific
hybridization of the probe and a PKD1 polynucleotide, thereby
detecting the presence of a mutant PKD1 polynucleotide sequence in
the sample.
[0023] The present invention further relates to a method of
identifying a subject having or is at risk of having a
PKD1-associated disorder. Such a method can be performed, for
example, by contacting nucleic acid molecules in a sample from a
subject with at least one primer pair of the invention under
conditions suitable for amplification of a PKD1 polynucleotide by
the primer pair, thereby generating an amplification product; and
testing an amplification product for the presence or absence of a
mutation indicative of a PKD1-associated disorder. As disclosed
herein, the absence of such a mutation identifies the subject as
not having or at risk of the having a PKD1-associated disorder,
wherein the presence of such a mutation identifies the subject as
having or is at risk of having a PKD1-associated disorder, for
example, ADPKD or acquired cystic disease.
[0024] A primer pair useful in a diagnostic method of the invention
can include at least one primer pair selected from SEQ ID NO:3 and
4; SEQ ID NO:5 and 6; SEQ ID NOS:7 and 8; SEQ ID NOS:9 and 10; SEQ
ID NOS:11 and 12; SEQ ID NOS:13 and 14; SEQ ID NOS:15 and 16; SEQ
ID NOS:17 and 18; and SEQ ID NOS:9 and 113. The subject can be any
subject having a PKD1 gene and susceptible to a PKD1-associated
disorder, including a vertebrate subject, and particularly a
mammalian subject such as a cat or a human. In addition, the
diagnostic method can be performed in a high throughput format,
thereby allowing the examination of a large number samples in a
cost-effective manner.
[0025] The diagnostic method can further include contacting the
amplification product generated as described above with at least a
second primer pair, under conditions suitable for nested
amplification of the amplification product by a second primer pair,
thereby generating a nested amplification product. The second
primer pair can be, for example, a primer pair selected from SEQ ID
NOS:19 and 20; SEQ ID NOS:21 and 22; SEQ ID NOS:23 and 24; SEQ ID
NOS:25 and 26; SEQ ID NOS:27 and 28; SEQ ID NOS:29 and 30; SEQ ID
NOS:31 and 32; SEQ ID NOS:33 and 34; SEQ ID NOS:35 and 36; SEQ ID
NOS:37 and 38; SEQ ID NOS:39 and 40; SEQ ID NOS:41 and 42; SEQ ID
NOS:43 and 44; SEQ ID NOS:45 and 46; SEQ ID NOS:47 and 48; SEQ ID
NOS:49 and 50; SEQ ID NOS:51 and 61; a primer pair formed using two
consecutive primers set forth in Table 2 as SEQ ID NOS:62 to 96,
113, and 97 to 112 (i.e., SEQ ID NOS: 62 and 63, SEQ ID NOS:64 and
65, and so on); and a combination thereof, in which case, the step
of testing the amplification product for the presence or absence of
a mutation comprises testing the nested amplification product. It
should be recognized that the selection of a primer pair for nested
amplification is based, in part, on the sequence of the
PKD1-specific amplification product that is to be used as a
template for the nested amplification, i.e., nested primer pairs
are selected such that they can hybridize to a target PKD1-specific
amplification product and can amplify the target sequence.
[0026] An amplification product can be tested for the presence or
absence of the mutation, for example, by determining the nucleotide
sequence of the amplification product, and comparing it to a
corresponding nucleotide sequence of SEQ ID NO:1; by determining
the melting temperature of the amplification product, and comparing
it to the melting temperature of a corresponding nucleotide
sequence of SEQ ID NO:1, for example, using a method such as
denaturing high performance liquid chromatography; or by contacting
the amplification product with an oligonucleotide probe containing
nucleotide 474 of SEQ ID NO:1, wherein nucleotide 474 is a T;
nucleotide 487 of SEQ ID NO:1, wherein nucleotide 487 is an A;
nucleotide 3110 of SEQ ID NO:1, wherein nucleotide 3110 is a C;
nucleotide 8298 of SEQ ID NO:1, wherein nucleotide 8298 is a G;
nucleotide 9164 of SEQ ID NO:1, wherein nucleotide 9164 is a G;
nucleotide 9213 of SEQ ID NO:1, wherein nucleotide 9213 is an A;
nucleotide 9326 of SEQ ID NO:1, wherein nucleotide 9326 is a T;
nucleotide 9367 of SEQ ID NO:1, wherein nucleotide 9367 is a T;
nucleotide 10064 of SEQ ID NO:1, wherein nucleotide 10064 is an A;
nucleotide 10143 of SEQ ID NO:1, wherein nucleotide 10143 is a G;
nucleotide 10234 of SEQ ID NO:1, wherein nucleotide 10234 is a C;
and nucleotide 10255 of SEQ ID NO:1, wherein nucleotide 10255 is a
T, under conditions suitable for selective hybridization of the
probe to a mutant PKD1 polypeptide, which can be a normal variant
or can be a mutant PKD1 polynucleotide associated with a
PKD1-associated disorder.
[0027] The present invention also relates to a method of diagnosing
a PKD1-associated disorder in a subject suspected of having a
PKD1-associated disorder. Such a method is performed by amplifying
a nucleic acid sequence in a sample obtained from the subject using
a primer pair suitable for PKD1-specific amplification of a PKD1
gene sequence, for example, a primer pair such as SEQ ID NO:3 and
4; SEQ ID NOS:5 and 6; SEQ ID NOS:7 and 8; SEQ ID NOS:9 and 10; SEQ
ID NOS:11 and 12; SEQ ID NOS:13 and 14; SEQ ID NOS:15 and 16; SEQ
ID NOS:17 and 18, or SEQ ID NOS:9 and 113, thereby obtaining a
PKD1-specific first amplification product; and detecting a mutation
of a PKD1 gene sequence in the PKD1-specific first amplification
product, wherein the mutation is indicative of a PKD1-associated
disorder, thereby diagnosing a PKD1-associated disorder in the
subject.
[0028] In one embodiment, the diagnostic method includes a step of
further amplifying the first amplification product using a second
set of primer pairs to obtain a nested amplification product; and
detecting a PKD1 gene mutation in the nested amplification product.
The second set of primer pairs can be any primer pairs useful for
amplifying the PKD1-specific first amplification product,
including, for example, the primer pairs exemplified by SEQ ID
NOS:19 and 20; SEQ ID NOS:21 and 22; SEQ ID NOS:23 and 24; SEQ ID
NOS:25 and 26; SEQ ID NOS:27 and 28; SEQ ID NOS:29 and 30; SEQ ID
NOS:31 and 32; SEQ ID NOS:33 and 34; SEQ ID NOS:35 and 36; SEQ ID
NOS:37 and 38; SEQ ID NOS:39 and 40; SEQ ID NOS:41 and 42; SEQ ID
NOS:43 and 44; SEQ ID NOS:45 and 46; SEQ ID NOS:47 and 48; SEQ ID
NOS:49 and 50; SEQ ID NOS:51 and 61; or any of the primer pairs
formed using consecutive primers set forth in Table 2 as SEQ ID
NOS:62 to 96, 113, and 97 to 112.
[0029] In another method, the diagnostic method includes a step of
contacting the PKD1-specific first amplification product or second
amplification product with a probe comprising an isolated
polynucleotide, or an oligonucleotide portion thereof, comprising a
mutant of SEQ ID NO:1, under conditions such that the probe can
selectively hybridize to a mutant PKD1 polynucleotide; and
detecting selective hybridization of the probe to the first
amplification product, thereby diagnosing a PKD1-associated
disorder in the subject. The probe can be, for example, an
oligonucleotide portion of SEQ ID NO:1 that includes one or more of
nucleotide 474 is a T; nucleotide 487 is an A; nucleotide 3110 is a
C; nucleotide 8298 is a G; nucleotide 9164 is a G; nucleotide 9213
is an A; nucleotide 9326 is a T; nucleotide 9367 is a T; nucleotide
10064 is an A; nucleotide 10143 is a G; nucleotide 10234 is a C; or
nucleotide 10255 is a T.
[0030] The present invention also relates to a method of detecting
the presence of a mutant PKD1 polypeptide in a sample. Such a
method can be performed, for example, by contacting a sample
suspected of containing a mutant PKD1 polypeptide with an antibody
that specifically binds to a mutant PKD1 polypeptide, under
conditions which allow the antibody to bind to the mutant PKD1
polypeptide and detecting specific binding of the antibody and the
mutant PKD1 polypeptide in the sample. The detection of an
immunocomplex of the antibody and a mutant PKD1 polypeptide, for
example, indicates the presence of a mutant PKD1 polypeptide in the
sample. In one embodiment, the method is performed by contacting a
tissue sample from a subject suspected of containing a PKD1
polypeptide with the antibody that specifically binds a mutant PKD1
polypeptide under conditions that allow the antibody interact with
a PKD1 polypeptide and detecting specific binding of the antibody
and the PKD1 polypeptide in the tissue.
[0031] The present invention further relates to a kit for detecting
a mutant PKD1 polynucleotide, which can be a variant PKD1
polynucleotide or a mutant PKD1 polynucleotide associated with a
PKD1-associated disorder. The kit can contain, for example, a
carrier means containing therein one or more containers wherein a
first container contains a nucleotide sequence useful for detecting
a wild type or mutant PKD1 polynucleotide. As such, a nucleotide
sequence useful in a kit of the invention can be an oligonucleotide
comprising at least ten contiguous nucleotides of SEQ ID NO:1,
including at least one of nucleotide 474, wherein nucleotide 474 is
a T; nucleotide 487, wherein nucleotide 487 is an A; nucleotide
3110, wherein nucleotide 3110 is a C; a position corresponding to
nucleotide 3336, wherein nucleotide 3336 is deleted; nucleotide
3707, wherein nucleotide 3707 is an A; nucleotide 4168, wherein
nucleotide 4168 is a T; nucleotide 4885, wherein nucleotide 4885 is
an A; nucleotide 5168, wherein nucleotide 5168 is a T; nucleotide
6058, wherein nucleotide 6058 is a T; nucleotide 6078, wherein
nucleotide 6078 is an A; nucleotide 6089, wherein nucleotide 6089
is a T; nucleotide 6195, wherein nucleotide 6195 is an A;
nucleotide 6326, wherein nucleotide 6326 is a T; a position
corresponding to nucleotides 7205 to 7211, wherein nucleotides 7205
to 7211 are deleted; nucleotide 7376, wherein nucleotide 7376 is a
C; a nucleotide sequence corresponding to nucleotides 7535 to 7536,
wherein a GCG nucleotide sequence is inserted between nucleotides
7535 and 7536; nucleotide 7415, wherein nucleotide 7415 is a T;
[0032] nucleotide 7433, wherein nucleotide 7433 is a T; nucleotide
7696, wherein nucleotide 7696 is a T; nucleotide 7883, wherein
nucleotide 7883 is a T; nucleotide 8021, wherein nucleotide 8021 is
an A; a nucleotide sequence corresponding to nucleotide 8159 to
8160, wherein nucleotides 8159 to 8160 are deleted; nucleotide
8298, wherein nucleotide 8298 is a G; nucleotide 9164, wherein
nucleotide 9164 is a G; nucleotide 9213, wherein nucleotide 9213 is
an A; nucleotide 9326, wherein nucleotide 9326 is a T; nucleotide
9367, wherein nucleotide 9367 is a T; nucleotide 10064, wherein
nucleotide 10064 is an A; [0033] nucleotide 10143, wherein
nucleotide 10143 is a G; nucleotide 10234, wherein nucleotide 10234
is a C; or nucleotide 10255, wherein nucleotide 10255 is a T. A
nucleotide sequence useful in a kit of the invention also can
comprise one or both primers of a primer pair, particularly at
least a forward primer and a reverse primer as set forth in SEQ ID
NOS: 3 to 18; and the kit can further include at least a second
primer pair, including a forward and reverse primer as set forth in
SEQ ID NOS: 19 to 51 and 61 to 113. In another aspect, the present
invention relates to a kit containing an antibody that specifically
binds to a mutant PKD1 polypeptide or peptide portion thereof.
BRIEF DESCRIPTION OF THE DRAWINGS
[0034] FIG. 1 is a schematic showing the genomic structure of the
PKD1 gene (SEQ ID NO:1) and the relative position of locus-specific
templates and primers.
[0035] FIG. 2 shows the relative position of the BPF6-BPR6
long-range PCR template and the much shorter PKD1-specific exon 28
product, 28F-BPR6. The dashed line below exon 28 identified the
long range PCR amplification product that resulted when BPF6, the
sequence of which is common to the PKD1 gene and to the homologs,
was used in combination with the homolog-specific primer,
BPR6HG.
DETAILED DESCRIPTION OF THE INVENTION
[0036] The present invention provides compositions and methods for
identifying polycystic kidney disease-associated protein-1 (PKD1)
gene variants and mutants, and for diagnosing PKD1-associated
disorders in a subject. Prior to the present disclosure, the
ability to selectively examine the entire PKD1 gene for mutations
was precluded due to the high sequence homology of the PKD1 gene
and the PKD1 gene homologs, including those present with the PKD1
gene on human chromosome 16. As disclosed herein, polynucleotide
sequences have now been developed that are useful as probes and
primers for examining the entire PKD1 gene. Accordingly, the
present invention provides polynucleotides, and oligonucleotide
portions thereof, of a PKD1 gene and of PKD1 gene mutants that are
useful for detecting PKD1 mutations, and that can be diagnostic of
a PKD1-associated disorder.
[0037] Autosomal dominant polycystic kidney disease (ADPKD)
exhibits a transmission pattern typical of autosomal dominant
inheritance, where typically each offspring of an affected
individual has a 50% chance of inheriting the causative gene.
Linkage studies indicated that a causative gene is present on the
short arm of chromosome 16, near the I globin cluster; this locus
was designated PKD1 (Reeders et al., Nature, 317:542, 1985.) Though
other PKD-associated genes exist (for example, PKD2), defects in
PKD1 appear to cause ADPKD in about 85-90% of affected families
(Parfrey et al., New Eng. J. Med. 323:1085-1090, 1990; Peters et
al., Contrib. Nephrol. 97:128-139, 1992).
[0038] The PKD1 gene has been localized to chromosomal position
16p13.3, specifically to an interval of approximately 600 kb
between the markers ATPL and CMM65 (D16S84). This region is rich in
CpG islands that often flank transcribed sequences; it has been
estimated that this interval contains at least 20 genes. The
precise location of the PKD1 gene was pinpointed by the finding of
an ADPKD family whose affected members carry a translocation that
disrupts a 14 kb RNA transcript associated with this region
(European PKD Consortium, Cell, 77:881, 1994).
[0039] The genomic structure of the PKD1 gene, which is illustrated
in FIG. 1 (SEQ ID NO:1; see Appendix A; see, also, GenBank
Accession No. L39891, which is incorporated herein by reference),
extends over approximately 50 kb, contains 46 exons, and is
bisected by two large polypyrimidine tracts of approximately 2.5 kb
and 0.5 kb, respectively, in introns 21 and 22 (indicated by " . .
. CCTCCTCCT . . . " in FIG. 1). The replicated portion of the gene,
which begins prior to the 5'UTR and is believed to end in exon 34
(FIG. 1; stippled region), covers approximately two thirds of the
5' end of the gene and is duplicated several times in a highly
similar, transcribed fashion elsewhere in the human genome (Germino
et al., Genomics 13:144-151, 1992; European Chromosome 16 Tuberous
Sclerosis Consortium, 1993, Cell 75:1305-1315). The encoded PKD1
polypeptide is shown as SEQ ID NO:2 (see Appendix A; see, also,
GenBank Accession No. P98161, which is incorporated herein by
reference). It should be recognized that SEQ ID NO:2 is not the
same amino acid sequence as that shown to be encoded by GenBank
Accession No. L39891 (see, also, GenBank AAB59488), presumably due
to errors in predicting the encoded PKD1 polypeptide from the PKD1
gene sequence. Instead, the wild type PKD1 polypeptide sequence is
shown in SEQ ID NO:2 (GenBank Accession No. P98161).
[0040] The present invention provides a PKD1 gene specific primer,
which can be one of a primer pair. A primer of the invention
includes a 5' region and adjacent PKD1-specific 3' region, wherein
the 5' region has a nucleotide sequence that can hybridize to a
PKD1 gene sequence or to a PKD1 gene sequence and a PKD1 gene
homolog sequence, and the 3' region has a nucleotide sequence that
selectively hybridizes only to a PKD1 gene sequence, and
particularly not to a PKD1 gene homolog sequence, except that a
primer of the invention does not have a sequence as set forth in
SEQ ID NO:11, SEQ ID NO:18, SEQ ID NO:52, or SEQ ID NO:60. Thus, a
primer of the invention can have a sequence as set forth in any of
SEQ ID NOS:3, 4, 5, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 19, 20,
21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,
38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 61, 62, 63,
64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80,
81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,
98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111,
112 and 113, as well as a sequence that is substantially identical
to any of SEQ ID NOS:3 to 51 and 61 to 113, provided the sequence
comprises a 5' region that can hybridize to a PKD1 gene sequence or
to a PKD1 gene sequence and a PKD1 gene homolog sequence, and a 3'
region that selectively hybridizes to a PKD1 gene sequence, but not
to a PKD1 gene homolog sequence; and provided the sequence is not
otherwise specifically excluded herein.
[0041] As disclosed herein, a primer of the invention can be
prepared by aligning SEQ ID NO:1 with the PKD1 gene homologs
contained in GenBank Accession Nos. AC002039, AC010488, AC040158,
AF320593 and AF320594 (each of which is incorporated herein by
reference; see, also, Bogdanova et al., Genomics 74:333-341, 2001,
which is incorporated herein by reference) and identifying regions
having potential sequence differences, then selecting as
PKD1-specific primers those sequences that match over at least
about ten nucleotides and that have a mismatch at or adjacent to
the 3' terminus of the matched regions (see Example 1; see, also,
Phakdeekitcharoen et al., supra, 2000). Such primers are referred
to as "PKD1-specific primers" because, while they can hybridize to
a PKD1 gene and a PKD1 gene homologue, an extension product only
can be generated upon hybridization to a PKD1 gene due to the
mismatch of one or more nucleotides in the 3' region when the
primer hybridizes to a PKD1 gene homologue. Confirmation that a
selected oligonucleotide is a PKD1-specific primer can be made
using methods as disclosed herein (Example 1) or otherwise known in
the art. For example, a simple and straightforward method for
determining that a primer is a PKD1-specific primer of the
invention is to perform a primer extension or an amplification
reaction using the putative PKD1-specific primer and templates
including a PKD1 gene sequence and PKD1 gene homolog sequences, and
detecting a single extension product or amplification product
generated from the PKD1 gene template, but not the PKD1 gene
homolog templates. Sequences identified as PKD1-specific primers
using this or another method can be confirmed by performing various
control experiments as described by Watnick et al. (supra, 1999),
for example, by comparing an amplification product obtained in a
cell having a PKD1 gene with the products, if any, produced using
the radiation hybrid cell line, 145.19, which lacks the PKD1 gene
but contains PKD1 gene homologs.
[0042] A nucleotide sequence suspected of being useful as a
PKD1-specific primer also can be compared against a human genomic
DNA database using, for example, a BLAST search or other algorithm,
to confirm that the nucleotide sequence meets the requirements of a
PKD1-specific primer as defined herein. For example, a putative
PKD1-specific primer can be examined at the National Center for
Biotechnology Information (NCBI), which can be accessed on the
world wide web, by selecting the "Blast" option, thereafter
selecting the "Search for short nearly exact matches," entering in
the sequence to be examined, and, using the default search
algorithms (word size 7), searching the "nr" database, which
include all non-redundant GenBank+EMBL+DDBJ+PDB sequences, but no
EST, SST, GSS or HTGS sequences; output can be restricted to
showing only the top ten matches.
[0043] In a PKD1-specific primer of the invention, the 5' region
contains at least about ten contiguous nucleotides, generally at
least about 12 nucleotides, and usually about 14 to 18 nucleotides.
In addition, the 3' region of the primer contains at least one 3'
terminal nucleotide, and can include a sequence of at least about 2
to 6 nucleotides, particularly about 2 to 4 nucleotides. Where the
3' region consists of a single 3' terminal nucleotide, the primer
is selected such that the 3' terminal nucleotide is identical to a
nucleotide that is 5' and adjacent to the nucleotide sequence of
the PKD1 gene to which the 5' region of the primer can hybridize,
and is different from a nucleotide that is 5' and adjacent to a
nucleotide sequence of the PKD1 homolog to which the 5' region of
the primer can hybridize, i.e., provides a mismatched nucleotide.
Where the 3' region of the PKD1-specific primer contains two or
more nucleotides, one or more of the nucleotides can be mismatched,
and the mismatched nucleotide can, but need not include the 3'
terminal nucleotide, provided that when the mismatched nucleotide
or nucleotides do not include the 3' terminal nucleotide, the
primer cannot be extended when hybridized to a PKD1 gene
homolog.
[0044] PKD1-specific primers of the invention are exemplified by
primers that can selectively hybridize to a nucleotide sequence
that flanks and is within about fifty nucleotides of a nucleotide
sequence of SEQ ID NO:1 selected from about nucleotides 2043 to
4209; nucleotides 17907 to 22489; nucleotides 22218 to 26363;
nucleotides 26246 to 30615; nucleotides 30606 to 33957; nucleotides
36819 to 37140; nucleotides 37329 to 41258; and nucleotides 41508
to 47320. A primer of the invention is exemplified by any of SEQ ID
NOS: 3 to 10, 12 to 17, 19 to 51, and 61 to 113, and can have a
sequence substantially identical to any of SEQ ID NOS:3 to 51 and
61 to 113, provided the sequence meets the requirements of a
PKD1-specific primer as disclosed herein, and provided the sequence
is not a sequence as set forth in any of SEQ ID NO:11, SEQ ID
NO:18, SEQ ID NO:52, and SEQ ID NO:60.
[0045] A primer is considered to be "substantially identical" to
any of SEQ ID NOS:3 to 51 and 61 to 113 if the primer has at least
about 80% or 85%, generally at least about 90%, usually at least
about 95%, and particularly at least about 99% sequence identity
with one of SEQ ID NOS:3 to 51 and 61 to 113, and has a 5' region
and adjacent PKD1-specific 3' region, wherein the 5' region has a
nucleotide sequence that can hybridize to a PKD1 gene sequence or
to a PKD1 gene sequence and a PKD1 gene homolog sequence, and the
3' region has a nucleotide sequence that selectively hybridizes
only to a PKD1 gene sequence, and particularly not to a PKD1 gene
homolog sequence, as defined herein, except that a primer of the
invention does not have a sequence as set forth in SEQ ID NO:11,
SEQ ID NO:18, SEQ ID NO:52, or SEQ ID NO:60. As such, a primer of
the invention can include one or a few, but no more than about four
or five, more or fewer nucleotide than a primer as set forth in SEQ
ID NOS:3 to 51 and 61 to 113, provided the primer meets the
functional requirements as defined herein.
[0046] The present invention also provides primer pairs. In one
embodiment, a primer pair of the invention comprising a forward and
reverse PKD1-specific primer as disclosed herein. As such, a primer
pair of the invention can amplify a portion of SEQ ID NO:1
including about nucleotides 2043 to 4209; nucleotides 17907 to
22489; nucleotides 22218 to 26363; nucleotides 26246 to 30615;
nucleotides 30606 to 33957; nucleotides 36819 to 37140; nucleotides
37329 to 41258; nucleotides 41508 to 47320; or a combination
thereof. In general, a primer pair of the invention can produce an
amplification product of about ten kilobases or shorter, generally
about 7500 bases or shorter, and particularly about six kilobases
or shorter. Primer pairs of the invention are exemplified by a
forward primer and a reverse primer selected from SEQ ID NOS:3 to
18, for example, by any of SEQ ID NOS:3 and 4; SEQ ID NOS:5 and 6;
SEQ ID NOS:7 and 8; SEQ ID NOS:9 and 10; SEQ ID NOS:11 and 12; SEQ
ID NOS:13 and 14; SEQ ID NOS:15 and 16; SEQ ID NOS:17 and 18; and
SEQ ID NOS:9 and 113, which can be used to produce PKD1-specific
amplification products of about 0.3 kilobases to about 5.8
kilobases.
[0047] As disclosed herein, a set of eight polymerase chain
reaction (PCR) primer pairs can be used to prepare PKD1-specific
amplification products that encompass all of the exons and their
flanking introns within the replicated region of the PKD1 gene. In
view of the disclosed nucleotide sequences of the primers and of
SEQ ID NO:1, it will be recognized that additional PCR primer pairs
useful for a preparing PKD1-specific first amplification product
can be based on the exemplified primers and primer pairs, but can
include one or few additional nucleotides (based on SEQ ID NO:1) at
one or both ends of the exemplified primers, or can have one or a
few nucleotides of an exemplified primer deleted, and their
usefulness can be determined by comparing an amplification product
generated using the derived or modified primer with a PKD1-specific
amplification product as disclosed herein. As such, a primer pair
based, for example, on SEQ ID NOS: 3 and 4 can be used to generate
a PKD-1 specific amplification product containing about nucleotides
2043 to 4209 of SEQ ID NO:2, where in reference to "about"
nucleotides 2043 to 4209 of SEQ ID NO:2 accounts for the disclosure
that a primer pair used for amplification can be identical or
substantially identical to SEQ ID NOS: 3 and 4.
[0048] Accordingly, the present invention provides primer pairs
comprising a forward primer and a reverse primer having nucleotide
sequences as set forth in SEQ ID NOS:3 to 18; primer pairs
exemplified by SEQ ID NOS:3 and 4; SEQ ID NOS:5 and 6; SEQ ID NOS:7
and 8; SEQ ID NOS:9 and 10; SEQ ID NOS:11 and 12; SEQ ID NOS:13 and
14; SEQ ID NOS:15 and 16; SEQ ID NOS:17 and 18; and SEQ ID NOS:9
and 113; and substantially identical primer pairs that comprise
primers based on or derived from the exemplified primers, such
primer pairs being useful for preparing a PKD1-specific
amplification product. The primer pairs shown as SEQ ID NOS: 9 and
10 and SEQ ID NOS: 11 and 12 have been described by
Phakdeekitcharoen et al. (supra, 2000), as have the PKD1 specific
amplification products generated using these primers.
[0049] It should be recognized that certain primers and certain
primer pairs exemplified herein are not considered to be
encompassed within the present invention. For example, the primer
set forth in SEQ ID NO:11 has been described in U.S. Pat. No.
6,017,717 (which is incorporated herein by reference; column 24,
SEQ ID NO:15); and the primer set forth in SEQ ID NO:18 has been
described by Watnick et al. (Hum. Mol. Genet. 6:1473-1481, 1997,
which is incorporated herein by reference; see page 1479; KG8R25),
and, therefore, neither of these primers is considered to be a
primer of the invention. Nevertheless, the primers set forth as SEQ
ID NOS: 11 and 18 can be encompassed within the primer pairs of the
invention, including within various disclosed and exemplified
primer pairs, for example, the primer pairs set forth as SEQ ID
NOS:11 and 12 and as SEQ ID NOS:17 and 18, as well as within
combinations of two or more primer pairs, for example, a
combination comprising SEQ ID NOS:11 and 12 and SEQ ID NOS:13 and
14.
[0050] The primers set forth in SEQ ID NO:9 and SEQ ID NO:10 have
been described by Watnick et al. (Am. J. Hum. Genet. 65:1561-1571,
1999, which is incorporated herein by reference) and, therefore,
can be specifically excluded from certain embodiments of the
invention, as desired, for example, as encompassed within the
primers of the invention. It should be recognized, however, that
the combination of SEQ ID NOS:9 and 10 as a primer pair is not
described by Watnick et al. (supra, 1999). SEQ ID NOS:49 to 51 and
61 to 105 also have been described by Watnick et al. (supra, 1999)
and, therefore, can be specifically excluded from certain
embodiments of the invention, as desired.
[0051] Except as provided herein, a primer of the invention is
exemplified by any of SEQ ID NOS:3 to 51 and 61 to 113, as well as
substantially identical oligonucleotide primers that are based on
or derived from SEQ ID NOS:3 to 51 and 61 to 113. It should be
recognized, however, that the primer set forth as SEQ ID NO:12 is
substantially similar to the primer designated TWR2 by Watnick et
al. (Mol. Cell 2:247-251, 1998, which is incorporated herein by
reference; page 250; 5'-GCAGGGTGAGCAGGTGGGGCCATCCTA-3; SEQ ID
NO:60), and that the primer set forth as SEQ ID NO:10 is
substantially identical to SEQ ID NO:13 in U.S. Pat. No. 6,071,717
(5'-AGGTCAACGTGGGCCTCCAAGTAGT-3; SEQ ID NO:52). As such, a primer
having the nucleotide sequence of SEQ ID NO:52 or of SEQ ID NO:60
is specifically excluded from the primers that otherwise would be
encompassed within the scope of primers that have a sequence
substantially identical to the sequence of the primer set forth as
SEQ ID NO:12 or SEQ ID NO:10, respectively.
[0052] The present invention also provides an isolated mutant PKD1
polynucleotide, or an oligonucleotide portion thereof comprising a
mutation as disclosed herein. As used herein, the term "isolated"
or "purified," when used in reference to a polynucleotide,
oligonucleotide, or polypeptide, means that the material is in a
form other than that in which it normally is found in nature. Thus,
where a polynucleotide or polypeptide occurs in a cell in nature,
an isolated polynucleotide or purified polypeptide can be one that
separated, at least in part, from the materials with which it is
normally associated. In general, an isolated polynucleotide or a
purified polypeptide is present in a form in which it constitutes
at least about 5 to 10% of a composition, usually 20% to 50% of a
composition, particularly about 50% to 75% of a composition, and
preferably about 90% to 95% or more of a composition. Methods for
isolating a polynucleotide or polypeptide are well known and
routine in the art.
[0053] As part of or following isolation, a polynucleotide can be
joined to other polynucleotides, such as DNA molecules, for
example, for mutagenesis studies, to form fusion proteins, or for
propagation or expression of the polynucleotide in a host. The
isolated polynucleotides, alone or joined to other polynucleotides,
such as vectors, can be introduced into host cells, in culture or
in whole organisms. Such polynucleotides, when introduced into host
cells in culture or in whole organisms, nevertheless are considered
"isolated" because they are not in a form in which they exist in
nature. Similarly, the polynucleotides, oligonucleotides, and
polypeptides can be present in a composition such as a media
formulation (solutions for introduction of polynucleotides,
oligonucleotides, or polypeptides, for example, into cells or
compositions or solutions for chemical or enzymatic reactions which
are not naturally occurring compositions) and, therein remain
isolated polynucleotides, oligonucleotides, or polypeptides within
the meaning of that term as it is employed herein. An isolated
polynucleotide can be a polynucleotide that is not immediately
contiguous with nucleotide sequences with which it is immediately
contiguous in a genome or other naturally occurring cellular DNA
molecule in nature. Thus, a recombinant polynucleotide, which can
comprise a polynucleotide incorporated into a vector, an
autonomously replicating plasmid, or a virus; or into the genomic
DNA of a prokaryote or eukaryote, which does not normally express a
PKD1 polypeptide.
[0054] As used herein, the term "polynucleotide" or
"oligonucleotide" or "nucleotide sequence" or the like refers to a
polymer of two or more nucleotides or nucleotide analogs. The
polynucleotide can be a ribonucleic acid (RNA) or deoxyribonucleic
acid (DNA) molecule, and can be single stranded or double stranded
DNA or RNA, or a double stranded DNA:RNA hybrid. A polynucleotide
or oligonucleotide can contain one or more modified bases, for
example, inosine or a tritylated base. The bonds linking the
nucleotides in a polymer generally are phosphodiester bonds, but
can be other bonds routinely used to link nucleotides including,
for example, phosphorothioate bonds, thioester bonds, and the like.
A polynucleotide also can be a chemically, enzymatically or
metabolically modified form.
[0055] As used herein, the term "mutant PKD1 polynucleotide" means
a nucleotide sequence that has one or a few nucleotide changes as
compared to the nucleotide sequence set forth as SEQ ID NO:1. The
nucleotide change can be a deletion, insertion or substitution, and
can be silent such that there is no change in the reading frame of
a polypeptide encoded by the PKD1 polynucleotide, or can be a
change that results in an amino acid change or in the introduction
of a STOP codon into the polynucleotide, or a change in a
nucleotide sequence involved in transcription or translation of the
PKD1 polynucleotide, for example, a change that results in altered
splicing of a PKD1 gene transcript into an mRNA (see Example 2). As
disclosed herein, a mutant PKD1 polynucleotide can be a polymorphic
variant, which, other than one or a few nucleotide changes with
respect to SEQ ID NO:1, encodes a PKD1 polypeptide and does not
correlate with a PKD1 associated disorder, particularly ADPKD, or
can be a mutant PKD1 polynucleotide that contains one or more
mutations that correlate with a PKD1 associated disorder such as
ADPKD (see Example 2).
[0056] For convenience of discussion and for use as a frame of
reference, the PKD1 nucleotide sequence set forth in SEQ ID NO:1 is
referred to as a "wild type PKD1 polynucleotide" or a "wild type
PKD1 gene" sequence, and, similarly, the polypeptide set forth as
SEQ ID NO:2 is referred to as a "wild type PKD1 polypeptide."
However, while the presence of the wild type PKD1 gene sequence
(i.e., SEQ ID NO:1) in an individual correlates to the absence of
ADPKD in the individual, it should be recognized that polymorphic
variants of SEQ ID NO:1 also are found in individuals that do not
exhibit ADPKD or other PKD1-associated disorder. The term
"variants" or "polymorphic variants" is used herein to refer to
mutant PKD1 polynucleotide sequences (with respect to SEQ ID NO:1)
that do not correlate with the signs or symptoms characteristic of
a PKD1 associated disorder such as ADPKD. Variant PKD1
polynucleotides include, for example, nucleotide substitutions that
do not result in a change in the encoded amino acid, i.e., silent
mutations, such as G4885A, in which the wild type and mutant codons
both encode a threonine (T1558T), and C6058T, in which the wild
type and mutant codons both encode a serine (S1949S; see Example 2;
see, also, Phakdeekitcharoen et al., supra, 2000); those that do
not segregate with the disease, or those that are found in a panel
of unaffected individuals. As such, it should be recognized that
the term "mutant PKD1 polynucleotide" broadly encompasses PKD1
variants, which do not correlate with a PKD1 associated disorder,
as well as mutant PKD1 polynucleotides that correlate or are
associated with a PKD1 associated disorder.
[0057] Examples of mutant PKD1 polynucleotide sequences, including
variant PKD1 polynucleotide sequence, include sequences
substantially as set forth in SEQ ID NO:1, but having a mutation at
nucleotide 474, wherein nucleotide 474 is a T; nucleotide 487,
wherein nucleotide 487 is an A; nucleotide 3110, wherein nucleotide
3110 is a C; a position corresponding to nucleotide 3336, wherein
nucleotide 3336 is deleted; nucleotide 3707, wherein nucleotide
3707 is an A; nucleotide 4168, wherein nucleotide 4168 is a T;
nucleotide 4885, wherein nucleotide 4885 is an A; nucleotide 5168,
wherein nucleotide 5168 is a T; nucleotide 6058, wherein nucleotide
6058 is a T; nucleotide 6078, wherein nucleotide 6078 is an A;
nucleotide 6089, wherein nucleotide 6089 is a T; nucleotide 6195,
wherein nucleotide 6195 is an A; nucleotide 6326, wherein
nucleotide 6326 is a T; a position corresponding to nucleotides
7205 to 7211, wherein nucleotides 7205 to 7211 are deleted;
nucleotide 7376, wherein nucleotide 7376 is a C; a nucleotide
sequence corresponding to nucleotides 7535 to 7536, wherein a GCG
nucleotide sequence is inserted between nucleotides 7535 and 7536;
nucleotide 7415, wherein nucleotide 7415 is a T; nucleotide 7433,
wherein nucleotide 7433 is a T; nucleotide 7696, wherein nucleotide
7696 is a T; nucleotide 7883, wherein nucleotide 7883 is a T;
nucleotide 8021, wherein nucleotide 8021 is an A; a nucleotide
sequence corresponding to nucleotide 8159 to 8160, wherein
nucleotides 8159 to 8160 are deleted; nucleotide 8298, wherein
nucleotide 8298 is a G; nucleotide 9164, wherein nucleotide 9164 is
a G; nucleotide 9213, wherein nucleotide 9213 is an A; nucleotide
9326, wherein nucleotide 9326 is a T; nucleotide 9367, wherein
nucleotide 9367 is a T; nucleotide 10064, wherein nucleotide 10064
is an A; nucleotide 10143, wherein nucleotide 10143 is a G;
nucleotide 10234, wherein nucleotide 10234 is a C; or nucleotide
10255, wherein nucleotide 10255 is a T; or a combination thereof
(see Example 2; see, also, Tables 3 and 4). Examples of a mutant
PKD1 polynucleotide of the invention also include a polynucleotide
that encodes a PKD1 polypeptide having substantially as set forth
in SEQ ID NO:2, but having an A88V, W967R, G1166S; V1956E; R1995H;
R2408C; D2604N; L2696R, R2985G, R3039C, V3285I, H3311R mutation, or
a combination thereof, as well as polypeptides that have, for
example, an addition of a Gly residue between amino acid residues
2441 and 2442 of SEQ ID NO:2 due to an insertion, or that terminate
with amino acid 3000 of SEQ ID NO:2 due to the presence of a STOP
codon at the position in SEQ ID NO:1 that would otherwise encode
amino acid 3001 (see, also, Table 4; Example 2).
[0058] Additional examples of mutant PKD1 polynucleotides of the
invention include polynucleotide sequences that selectively
hybridize to the complements of the polynucleotide sequences, or
oligonucleotide portions thereof, as disclosed herein, under highly
stringent hybridization conditions, e.g., hybridization to
filter-bound DNA in 0.5M NaHPO.sub.4, 7% sodium dodecyl sulfate
(SDS), 1 mM EDTA at 65.degree. C., and washing in
0.1.times.SSC/0.1% SDS at 68.degree. C. (Ausubel et al., Current
Protocols in Molecular Biology, (Green Publishing Associates, Inc.,
and John Wiley & Sons, Inc., New York 1989), and supplements;
seep. 2.10.3; Sambrook et al., Molecular Cloning: A laboratory
manual (Cold Spring Harbor Laboratory Press, 1989), which are
incorporated herein by reference), as well as polynucleotides that
encode a PKD1 polypeptide substantially as set forth in SEQ ID
NO:2, but having one or more mutations; or an RNA corresponding to
such a polynucleotide.
[0059] A polynucleotide or polypeptide sequence that is
"substantially identical" to a PKD1 polynucleotide of SEQ ID NO:1
or a polypeptide sequence of SEQ ID NO:2 generally is at least 80%
or 85%, usually at least about 90%, and particularly at least about
95%, and preferably at least about 99% identical to the nucleotide
sequence or amino acid sequence as set forth in SEQ ID NO:1 or SEQ
ID NO:2, respectively. It should be recognized, however, that a
mutation in a PKD1 gene sequence can result in the expression of a
truncated PKD1 polypeptide, or even a complete loss of expression
of the PKD1 polypeptide. As such, while a mutant PKD1
polynucleotide is identified as being substantially identical to
SEQ ID NO:1, it may not always be possible to make the same
comparison with respect to the encoded polypeptides. In one aspect
of the invention, a polynucleotide or polypeptide sequence that is
substantially identical to SEQ ID NO:1 or 2 will vary at one or
more sites having a mutation, for example, a mutation present in a
mutant PKD1 polynucleotide as set forth in the preceding paragraph.
Sequence identity can be measured using sequence analysis software
(e.g., Sequence Analysis Software Package of the Genetics Computer
Group, University of Wisconsin Biotechnology Center, 1710
University Avenue, Madison Wis. 53705).
[0060] A polynucleotide or oligonucleotide portion thereof of the
invention can be useful, for example, as a probe or as a primer for
an amplification reaction. Reference to an "oligonucleotide
portion" of a mutant PKD1 polynucleotide means a nucleotide
sequence of the mutant PKD1 polynucleotide that is less than the
full length polynucleotide. Generally, a polynucleotide useful as a
probe or a primer contains at least about 10 nucleotides, and
usually contains about 15 to 30 nucleotides or more (see, for
example, Tables 1 and 2). Polynucleotides can be prepared by any
suitable method, including, for example, by restriction enzyme
digestion of an appropriate polynucleotide, by direct chemical
synthesis using a method such as the phosphotriester method (Narang
et al., 1979, Meth. Enzymol., 68:90-99); the phosphodiester method
(Brown et al., 1979, Meth. Enzymol., 68:109-151); the
diethylphosphoramidite method (Beaucage et al., 1981, Tetrahedron
Lett., 22:1859-1862); the triester method (Matteucci et al., 1981,
J. Am. Chem. Soc., 103:3185-3191), including by automated synthesis
methods; or by a solid support method (see, for example, U.S. Pat.
No. 4,458,066). In addition, a polynucleotide or oligonucleotide
can be prepared using recombinant DNA methods as disclosed herein
or otherwise known in the art.
[0061] An oligonucleotide of the invention can include a portion of
a mutant PKD1 polynucleotide, including, for example, a sequence
substantially identical to that of SEQ ID NO:1, except wherein
nucleotide 474 is a T; or wherein nucleotide 487 is an A; or
wherein nucleotide 3110 is a C; or wherein nucleotide 8298 is a G;
or wherein nucleotide 9164 is a G; or wherein nucleotide 9213 is an
A; or wherein nucleotide 9326 is a T; or wherein nucleotide 9367 is
a T; or wherein nucleotide 10064 is an A; or wherein nucleotide
10143 is a G; or wherein nucleotide 10234 is a C; or wherein
nucleotide 10255 is a T; or wherein the oligonucleotide contains a
combination of such substitutions with respect to SEQ ID NO:1.
Thus, as disclosed herein, the oligonucleotide can be any length
and can encompass one or more of the above mutations.
[0062] An oligonucleotide of the invention can selectively
hybridize to a mutant PKD1 polynucleotide sequence as disclosed
herein. As such, the oligonucleotide does not hybridize
substantially, if at all, to a wild type PKD1 polynucleotide (i.e.,
to SEQ ID NO:1). As used herein, the term "selectively hybridize"
refers to the ability of an oligonucleotide (or polynucleotide)
probe to hybridize to a selected sequence, but not to a highly
related nucleotide sequence. For example, a oligonucleotide of the
invention selectively hybridizes to a mutant PKD1 polynucleotide,
but not substantially to a corresponding sequence of SEQ ID NO:1.
As such, hybridization of the oligonucleotide to SEQ ID NO:1
generally is not above background, or, if some hybridization
occurs, is at least about ten-fold less than the amount of
hybridization that occurs with respect to the mutant PKD1
polynucleotide.
[0063] In addition, the term "hybridize" is used herein to have its
commonly understood meaning of two nucleotide sequences that can
associate due to shared complementarity. As disclosed herein, a
primer of the invention can hybridize to PDK1 gene and may also
hybridize to a PDK1 gene homolog, but generally does not
substantially hybridize to a nucleotide sequence other than a PKD1
gene or PKD1 gene homolog. Desired hybridization conditions,
including those that allow for selective hybridization, can be
obtained by varying the stringency of the hybridization conditions,
based, in part, on the length of the sequences involved, the
relative G:C content, the salt concentration, and the like (see
Sambrook et al., supra, 1989). Hybridization conditions that are
highly stringent conditions are used for selective hybridization
and can be used for hybridization of a primer or primer pair of the
invention to a PKD1 gene or PKD1 gene homolog, and include, for
example, washing in 6.times.SSC/0.05% sodium pyrophosphate at about
37.degree. C. (for 14 nucleotide DNA probe), about 48.degree. C.
(for 17 nucleotide probe), about 55.degree. C. (for a 20 nucleotide
probe), and about 60.degree. C. (for a 23 nucleotide probe). As
disclosed herein, polynucleotides that selectively hybridize to a
mutant PKD1 polynucleotide provide a means to distinguish the
mutant PKD1 polynucleotide from a wild type PKD1
polynucleotide.
[0064] A polynucleotide or oligonucleotide of the invention can be
used as a probe to screen for a particular PKD1 variant or mutant
of interest. In addition, the oligonucleotides of the invention
include a PKD1 antisense molecule, which can be useful, for
example, in PKD1 polynucleotide regulation and amplification
reactions of PKD1 polynucleotide sequences, including mutant PKD1
polynucleotide sequences. Further, such oligonucleotides can be
used as part of ribozyme or triple helix sequence for PKD1 gene
regulation. Still further, such oligonucleotides can be used as a
component of diagnostic method, whereby the level of PKD1
transcript can be determined or the presence of an ADPKD-causing
allele can be detected. Further, such oligonucleotides can be used,
for example, to screen for and identify PKD1 homologs from other
species.
[0065] The term "primer" or "PCR primer" refers to an isolated
natural or synthetic oligonucleotide that can act as a point of
initiation of DNA synthesis when placed under conditions suitable
for primer extension. Synthesis of a primer extension product is
initiated in the presence of nucleoside triphosphates and a
polymerase in an appropriate buffer at a suitable temperature. A
primer can comprise a plurality of primers, for example, where
there is some ambiguity in the information regarding one or both
ends of the target region to be synthesized. For instance, if a
nucleic acid sequence is determined from a protein sequence, a
primer generated to synthesize nucleic acid sequence encoding the
protein sequence can comprise a collection of primers that contains
sequences representing all possible codon variations based on the
degeneracy of the genetic code. One or more of the primers in this
collection will be homologous with the end of the target sequence
or a sequence flanking a target sequence. Likewise, if a conserved
region shows significant levels of polymorphism in a population,
mixtures of primers can be prepared that will amplify adjacent
sequences.
[0066] During PCR amplification, primer pairs flanking a target
sequence of interest are used to amplify the target sequence. A
primer pair typically comprises a forward primer, which hybridizes
to the 5' end of the target sequence, and a reverse primer, which
hybridizes to the 3' end of the target sequence. Except as
otherwise provided herein, primers of the present invention are
exemplified by those having the sequences set forth as SEQ ID NOS:3
to 51 and 61 to 113 (see Tables 1 and 2). Forward primers are
exemplified by SEQ ID NOS:3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23,
25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47 and 49; and reverse
primers are exemplified by SEQ ID NOS:4, 6, 8, 10, 12, 14, 16, 18,
20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, and 50.
A primer pair of the invention includes at least one forward primer
and at least one reverse primer that allows for generation of an
amplification product, which can be a long range PKD1-specific
amplification product or a nested amplification product of such an
amplification product, including a forward and reverse primer as
set forth in SEQ ID NOS:3 to 18 and of SEQ ID NOS:19 to 51 and 61
to 113, provided that the forward primer is 5' (or upstream) of the
reverse primer with reference to a target polynucleotide sequence,
and that the primers are in sufficient proximity such that an
amplification product can be generated.
[0067] Nucleic acid sequences that encode a fusion protein can be
produced and can be operatively linked to expression control
sequences. Such fusion proteins and compositions are useful in the
development of antibodies or to generate and purify peptides and
polypeptides of interest. As used herein, the term "operatively
linked" refers to a juxtaposition, wherein the components so
described are in a relationship permitting them to function in
their intended manner. For example, an expression control sequence
operatively linked to a coding sequence is ligated such that
expression of the coding sequence is achieved under conditions
compatible with the expression control sequences, whereas two
operatively linked coding sequences can be ligated such that they
are in the same reading frame and, therefore, encode a fusion
protein.
[0068] As used herein, the term "expression control sequences"
refers to nucleic acid sequences that regulate the expression of a
nucleic acid sequence to which it is operatively linked. Expression
control sequences are operatively linked to a nucleic acid sequence
when the expression control sequences control and regulate the
transcription and, as appropriate, translation of the nucleic acid
sequence. Thus, expression control sequences can include
appropriate promoters, enhancers, transcription terminators, a
start codon (i.e., ATG) in front of a protein-encoding gene,
splicing signals for introns, maintenance of the correct reading
frame of that gene to permit proper translation of the mRNA, and
STOP codons. Control sequences include, at a minimum, components
whose presence can influence expression, and can also include
additional components whose presence is advantageous, for example,
leader sequences and fusion partner sequences. Expression control
sequences can include a promoter.
[0069] A polynucleotide of the invention can comprise a portion of
a recombinant nucleic acid molecule, which, for example, can encode
a fusion protein. The polynucleotide, or recombinant nucleic acid
molecule, can be inserted into a vector, which can be an expression
vector, and can be derived from a plasmid, a virus or the like. The
expression vector generally contains an origin of replication, a
promoter, and one or more genes that allow phenotypic selection of
transformed cells containing the vector. Vectors suitable for use
in the present invention include, but are not limited to the
T7-based expression vector for expression in bacteria (Rosenberg et
al., Gene 56:125, 1987), the pMSXND expression vector for
expression in mammalian cells (Lee and Nathans, J. Biol. Chem.
263:3521, 1988); baculovirus-derived vectors for expression in
insect cells; and the like.
[0070] The choice of a vector will also depend on the size of the
polynucleotide sequence and the host cell to be employed in the
methods of the invention. Thus, the vector used in the invention
can be plasmids, phages, cosmids, phagemids, viruses (e.g.,
retroviruses, parainfluenzavirus, herpesviruses, reoviruses,
paramyxoviruses, and the like), or selected portions thereof (e.g.,
coat protein, spike glycoprotein, capsid protein). For example,
cosmids and phagemids are typically used where the specific nucleic
acid sequence to be analyzed or modified is large because these
vectors are able to stably propagate large polynucleotides. Cosmids
and phagemids are particularly suited for the expression or
manipulation of the PKD1 polynucleotide of SEQ ID NO:1 or a mutant
PKD1 polynucleotide.
[0071] In yeast, a number of vectors containing constitutive or
inducible promoters can be used (see Ausubel et al., supra, 1989;
Grant et al., Meth. Enzymol. 153:516-544, 1987; Glover, DNA
Cloning, Vol. II, IRL Press, Washington D.C., Ch. 3, 1986; and
Bitter, Meth. Enzymol. 152:673-684, 1987; and The Molecular Biology
of the Yeast Saccharomyces, Eds. Strathern et al., Cold Spring
Harbor Press, Vols. I and II, 1982). A constitutive yeast promoter
such as ADH or LEU2 or an inducible promoter such as GAL can be
used ("Cloning in Yeast," Ch. 3, Rothstein, In "DNA Cloning" Vol.
11, A Practical Approach, ed. Glover, IRL Press, 1986).
Alternatively, vectors can be used which promote integration of
foreign DNA sequences into the yeast chromosome. The construction
of expression vectors and the expression of genes in transfected
cells involves the use of molecular cloning techniques also well
known in the art (see Sambrook et al., supra, 1989; Ausubel et al.,
supra, 1989). These methods include in vitro recombinant DNA
techniques, synthetic techniques and in vivo recombination/genetic
recombination.
[0072] A polynucleotide or oligonucleotide can be contained in a
vector and can be introduced into a cell by transformation or
transfection of the cell. By "transformation" or "transfection" is
meant a permanent (stable) or transient genetic change induced in a
cell following incorporation of new DNA (i.e., DNA exogenous to the
cell). Where the cell is a mammalian cell, a permanent genetic
change is generally achieved by introduction of the DNA into the
genome of the cell.
[0073] A transformed cell or host cell can be any prokaryotic or
eukaryotic cell into which (or into an ancestor of which) has been
introduced, by means of recombinant DNA techniques, a
polynucleotide sequence of the invention or fragment thereof.
[0074] Transformation of a host cell can be carried out by
conventional techniques as are well known to those skilled in the
art. Where the host is prokaryotic, such as E. coli, competent
cells which are capable of DNA uptake can be prepared from cells
harvested after exponential growth phase and subsequently treated
by the CaCl.sub.2 method by procedures well known in the art, or
using MgCl.sub.2 or RbCl. Transformation can also be performed
after forming a protoplast of the host cell or by
electroporation.
[0075] When the host is a eukaryote, such methods of transfection
include the use of calcium phosphate co-precipitates, conventional
mechanical procedures such as microinjection, electroporation,
insertion of a plasmid encased in liposomes, or the use of virus
vectors, or other methods known in the art. One method uses a
eukaryotic viral vector, such as simian virus 40 (SV40) or bovine
papillomavirus, to transiently infect or transform eukaryotic cells
and express the protein. (Eukaryotic Viral Vectors, Cold Spring
Harbor Laboratory, Gluzman ed., 1982). Preferably, a eukaryotic
host is utilized as the host cell as described herein. The
eukaryotic cell can be a yeast cell (e.g., Saccharomyces
cerevisiae), or can be a mammalian cell, including a human
cell.
[0076] A variety of host-expression vector systems can be utilized
to express a PKD1 polynucleotide sequence such as SEQ ID NO:1, a
coding sequence of SEQ ID NO:1 or a mutant PKD1 polynucleotide.
Such host-expression systems represent vehicles by which the
nucleotide sequences of interest can be produced and subsequently
purified, and also represent cells that, when transformed or
transfected with the appropriate nucleotide coding sequences, can
express a PKD1 protein, including a PKD1 variant or mutant
polypeptide or peptide portion thereof in situ. Such cells include,
but are not limited to, microorganisms such as bacteria (e.g., E.
coli, B. subtilis) transformed with recombinant bacteriophage DNA,
plasmid DNA or cosmid DNA expression vectors containing a PKD1
polynucleotide, or oligonucleotide portion thereof (wild type,
variant or other mutant); yeast (e.g., Saccharomyces, Pichia)
transformed with recombinant yeast expression vectors containing a
PKD1 polynucleotide, or oligonucleotide portions thereof (wild
type, variant or other PKD1 mutant); insect cell systems infected
with recombinant virus expression vectors (e.g., baculovirus)
containing a PKD1 polynucleotide, or oligonucleotide portion
thereof (wild type, PKD1 variant or other mutant); plant cell
systems infected with recombinant virus expression vectors (e.g.,
cauliflower mosaic virus or tobacco mosaic virus) or transformed
with recombinant plasmid expression vectors (e.g., Ti plasmid)
containing a mutant PKD1 polynucleotide, or oligonucleotide portion
thereof; or mammalian cell systems (e.g., COS, CHO, BHK, 293, 3T3)
harboring recombinant expression constructs containing promoters
derived from the genome of mammalian cells (e.g., metallothionein
promoter) or from mammalian viruses (e.g., the adenovirus late
promoter; the vaccinia virus 7.5K promoter).
[0077] In bacterial systems, a number of expression vectors can be
advantageously selected depending upon the use intended for the
PKD1 protein (wild type, variant or other PKD1 mutant) being
expressed. For example, when a large quantity of such a protein is
to be produced, for the generation of antibodies, which can be used
to identify or diagnose PKD1-associated diseases or disorders, or
to screen peptide libraries, vectors that direct the expression of
high levels of fusion protein products that are readily purified
can be desirable. Such vectors include, but are not limited to, the
E. coli expression vector pUR278 (Ruther et al., 1983, EMBO J.
2:1791), in which a PKD1 polynucleotide, or oligonucleotide portion
thereof (wild type, variant or other mutant) can be ligated
individually into the vector in frame with the lac Z coding region
so that a fusion protein is produced; pIN vectors (Inouye and
Inouye, Nucl. Acids Res. 13:3101-3109, 1985; Van Heeke and
Schuster, J. Biol. Chem. 264:5503-5509, 1989); and the like. pGEX
vectors can also be used to express foreign polypeptides as fusion
proteins with glutathione S-transferase (GST). In general, such
fusion proteins are soluble and can easily be purified from lysed
cells by adsorption to glutathione-agarose beads followed by
elution in the presence of free glutathione. The pGEX vectors are
designed to include thrombin or factor Xa protease cleavage sites
so that the cloned PKD1 protein, variant or mutant can be released
from the GST moiety.
[0078] In an insect system, Autographa californica nuclear
polyhedrosis virus (AcNPV) is used as a vector to express foreign
genes. The virus grows in Spodoptera frugiperda cells. A PKD1
polynucleotide, or oligonucleotide portion thereof can be cloned
individually into non-essential regions (for example the polyhedrin
gene) of the virus and placed under control of an AcNPV promoter
(for example the polyhedrin promoter). Successful insertion of a
PKD1 polynucleotide, or oligonucleotide portion thereof will result
in inactivation of the polyhedrin gene and production of
non-occluded recombinant virus (i.e., virus lacking the
proteinaceous coat coded for by the polyhedrin gene). These
recombinant viruses are then used to infect Spodoptera frugiperda
cells in which the inserted gene is expressed (see Smith et al.,
1983, J. Virol. 46:584; U.S. Pat. No. 4,215,051).
[0079] In mammalian host cells, a number of viral-based expression
systems can be utilized. In cases where an adenovirus is used as an
expression vector, a PKD1 polynucleotide, or oligonucleotide
portion thereof, can be ligated to an adenovirus
transcription/translation control complex, e.g., the late promoter
and tripartite leader sequence. This chimeric gene can then be
inserted in the adenovirus genome by in vitro or in vivo
recombination. Insertion in a non-essential region of the viral
genome such as the E1 or E3 region results in a recombinant virus
that is viable and capable of expressing a PKD1 protein (e.g.,
wild-type, variants or mutants thereof) in infected hosts (Logan
and Shenk, Proc. Natl. Acad. Sci., USA 81:3655-3659, 1984).
Specific initiation signals can also be required for efficient
translation of an inserted PKD1 sequence. These signals include the
ATG initiation codon and adjacent sequences. Where an entire PKD1
polynucleotide, including its own initiation codon and adjacent
sequences, is inserted into the appropriate expression vector, no
additional translational control signals can be needed. However,
where only a portion of a PKD1 sequence is inserted, exogenous
translational control signals, including, for example, an ATG
initiation codon, must be provided. Furthermore, the initiation
codon must be in phase with the reading frame of the desired coding
sequence to ensure translation of the entire insert. These
exogenous translational control signals and initiation codons can
be of a variety of origins, both natural and synthetic. The
efficiency of expression can be enhanced by the inclusion of
appropriate transcription enhancer elements, transcription
terminators, and the like (see Bittner et al., Meth. Enzymol.
153:516-544, 1987).
[0080] In addition, a host cell strain can be chosen which
modulates the expression of the inserted sequences, or modifies and
processes the expressed polypeptide in a specific fashion. Such
modifications (e.g., glycosylation) and processing (e.g., cleavage)
of protein products can be important for the function of the
protein. Different host cells have characteristic and specific
mechanisms for the post-translational processing and modification
of proteins. Appropriate cell lines or host systems can be chosen
to ensure the correct modification and processing of the foreign
protein being expressed. To this end, eukaryotic host cells which
possess the cellular machinery for proper processing of the primary
transcript, glycosylation, and phosphorylation of the polypeptide
can be used. Such mammalian host cells include, but are not limited
to, CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3, WI38, and the
like.
[0081] For long term, high yield production of recombinant
proteins, stable expression is preferred. For example, cell lines
that stably express a PKD1 protein, including wild-type, variants
or mutants of PKD1, can be engineered. Rather than using expression
vectors which contain viral origins of replication, host cells can
be transformed with DNA controlled by appropriate expression
control elements (e.g., promoter and/or enhancer sequences,
transcription terminators, polyadenylation sites, and the like),
and a selectable marker. Following the introduction of the foreign
DNA, engineered cells can be grown for 1-2 days in an enriched
media, then switched to selective media. The selectable marker in
the recombinant plasmid confers resistance to the selection and
allows cells to stably integrate the plasmid into their chromosomes
and grow to form foci, which can be cloned and expanded into cell
lines. This method can advantageously be used to engineer cell
lines that express a PKD1 variant or mutant polypeptide. Such
engineered cell lines can be particularly useful in screening and
evaluation of compounds that affect the endogenous activity of a
variant or mutant PKD1 polypeptide. Such engineered cell lines also
can be useful to discriminate between factors that have specific
vs. non-specific effects. In particular, mutant cell lines should
lack key functions, and various mutations can be used to identify
key functional domains using in vivo assays.
[0082] A number of selection systems can be used, including but not
limited to the herpes simplex virus thymidine kinase (Wigler et
al., Cell 11:223, 1977), hypoxanthine-guanine
phosphoribosyltransferase (Szybalska and Szybalski, Proc. Natl.
Acad. Sci. USA 48:2026, 1962), and adenine
phosphoribosyltransferase (Lowy et al., Cell 22:817, 1980) genes
can be employed in tk.sup.-, hgprt.sup.- or aprt.sup.- cells,
respectively. Also, antimetabolite resistance can be used as the
basis of selection for dhfr, which confers resistance to
methotrexate (Wigler et al., Proc. Natl. Acad. Sci. USA 77:3567,
1980; O'Hare et al., Proc. Natl. Acad. Sci. USA 78:1527, 1981);
gpt, which confers resistance to mycophenolic acid Mulligan and
Berg, Proc. Natl. Acad. Sci. USA 78:2072, 1981); neo, which confers
resistance to the aminoglycoside G-418 (Colberre-Garapin et al., J.
Mol. Biol. 150:1, 1981); and hygro, which confers resistance to
hygromycin (Santerre et al., Gene 30:147, 1984) genes. Accordingly,
the invention provides a vector that contains a mutant PKD1
polynucleotide, or oligonucleotide portion thereof, or one or more
primers or their complements, including an expression vector that
contains any of the foregoing sequences operatively associated with
a regulatory element that directs the expression of a coding
sequence or primer; and also provides a host cell that contains any
of the foregoing sequences, alone or operatively associated with a
regulatory element, which can directs expression of a polypeptide
encoded the polynucleotide, as appropriate.
[0083] In addition to mutant PKD1 polynucleotide sequences
disclosed herein, homologs of mutant PKD1 polynucleotide of the
invention, including a non-human species, can be identified and
isolated by molecular biological techniques well known in the art.
Further, mutant PKD1 alleles and additional normal alleles of the
human PKD1 polynucleotide, can be identified using the methods of
the invention. Still further, there can exist genes at other
genetic loci within the human genome that encode proteins having
extensive homology to one or more domains of the PKD1 polypeptide
(SEQ ID NO:2). Such genes can also be identified including
associated variants and mutants by the methods of the
invention.
[0084] A homolog of a mutant PKD1 polynucleotide sequence can be
isolated by performing a polymerase chain reaction (PCR; see U.S.
Pat. No. 4,683,202, which is incorporated herein by reference)
using two oligonucleotide primers, which can be selected, for
example, from among SEQ ID NOS:3 to 51, preferably from among SEQ
ID NOS: 3 to 18, or can be degenerate primer pools designed on the
basis of the amino acid sequences of a PKD1 polypeptide such as
that set forth in SEQ ID NO:2 or a mutant thereof as disclosed
herein. The template for the reaction can be cDNA obtained by
reverse transcription of mRNA prepared from human or non-human cell
lines or tissue known to express a PKD1 allele or PKD1 homologue.
The PCR product can be subcloned and sequenced or manipulated in
any number of ways (e.g., further manipulated by nested PCR) to
insure that the amplified sequences represent the sequences of a
PKD1 or a PKD mutant polynucleotide sequence. The PCR fragment can
then be used to isolate a full length PKD1 cDNA clone (including
clones containing a mutant PKD1 polynucleotide sequence) by
labeling the amplified fragment and screening a nucleic acid
library (e.g., a bacteriophage cDNA library). Alternatively, the
labeled fragment can be used to screen a genomic library (for
review of cloning strategies, see, for example, Sambrook et al.,
supra, 1989; Ausubel et al., supra, 1989).
[0085] The present invention also provides a purified mutant PKD1
polypeptide, or a peptide portion thereof. As disclosed herein, a
mutant PKD1 polypeptide has an amino acid sequence substantially
identical to SEQ ID NO:2, and includes a mutation resulting in the
deletion, addition (insertion), or substitution of an amino acid of
SEQ ID NO:2, or is truncated with respect to SEQ ID NO:2. Examples
of such mutations include, with respect to SEQ ID NO:2, an A88V,
W967R, G1166S; V1956E; R1995H; R2408C; D2604N; L2696R, R2985G,
R3039C, V3285I, or H3311R mutation, an addition of a Gly residue
between amino acid residues 2441 and 2442 of SEQ ID NO:2 due to an
insertion, or a truncated PKD1 polypeptide terminates with amino
acid 3000 of SEQ ID NO:2 due to the presence of a STOP codon at the
position in SEQ ID NO:1 that would otherwise encode amino acid
3001; as well as mutant PKD1 polypeptides having a combination of
such mutations (see Table 4).
[0086] A mutant PKD1 polypeptide or peptide portion thereof can
contain one or more of the exemplified mutations. As used herein,
reference to a peptide portion of SEQ ID NO:2 or of a mutant PKD1
polypeptide refers to a contiguous amino acid sequence of SEQ ID
NO:2 or of SEQ ID NO:2 including a mutation as disclosed herein,
respectively, that contains fewer amino acids than full length wild
type PKD1 polypeptide. Generally, a peptide portion of a PKD1
polypeptide or a mutant PKD1 polypeptide contains at least about
five amino acids (or amino acid derivatives or modified amino
acids), each linked by a peptide bond or a modified form thereof,
usually contains at least about eight amino acids, particularly
contains about ten amino acids, and can contain twenty or thirty or
more amino acids of SEQ ID NO:2. In particular, where the peptide
is a peptide portion of a mutant PKD1 polypeptide, the peptide
includes a mutant amino acid with respect to SEQ ID NO:2.
[0087] The mutant PKD1 polypeptides and peptide fragments thereof
of the invention include a PKD1 polypeptide or peptide having a
sequence substantially identical to that set forth in SEQ ID NO:2,
and having one or a combination of the following mutations: A88V,
W967R, L2696R, R2985G, R3039C, V3285I, or H3311R, or a mutation
resulting in termination of the mutant PKD1 polypeptide at amino
acid 3000 (with respect to SEQ ID NO:2) due to the presence of a
STOP codon at the position that otherwise would encode amino acid
3001. The wild type PKD1 polypeptide (SEQ ID NO:2) contains 4303
amino acid residues and has a predicted molecular mass of
approximately 467 kilodaltons (kDa). Further encompassed by the
present invention are mutant PKD1 polypeptides that are truncated
with respect to SEQ ID NO:2, for example, a mutation of SEQ ID NO:1
resulting in a G9213A, which results in premature termination of
the encoded PKD1 polypeptide (see Example 2). Such truncated
products can be associated with PKD1-associated disorders such as
ADPKD (see, also, Table 4).
[0088] PKD1 polypeptides that are functionally equivalent to a wild
type PKD1 polypeptide, including variant PKD1 polypeptides, which
can contain a deletion, insertion or substitution of one or more
amino acid residues with respect to SEQ ID NO:2, but that
nevertheless result in a phenotype that is indistinguishable from
that conferred by SEQ ID NO:2, are encompassed within the present
invention. Such amino acid substitutions, for example, generally
result in similarity in polarity, charge, solubility,
hydrophobicity, hydrophilicity, amphipatic nature or the like of
the residues involved. For example, negatively charged amino acids
include aspartic acid and glutamic acid; positively charged amino
acids include lysine and arginine; amino acids with uncharged polar
head groups having similar hydrophilicity values include the
following: leucine, isoleucine, valine, glycine, alanine,
asparagine, glutamine, serine, threonine, phenylalanine and
tyrosine. In many cases, however, a nucleotide substitution can be
silent, resulting in no change in the encoded PKD1 polypeptide (see
Example 2). Such variant PKD1 polynucleotides are exemplified by
those encoded by the variant PKD1 polynucleotide sequences
substantially identical to SEQ ID NO:1 (SEQ ID NO:2), but
containing (encoding) G487A (A92A), C9367T (G3052G), T10234C
(L3341L), and G10255T (R3348R) as shown in Table 3 (see, also,
Example 2), and by C9494T (L3095L).
[0089] Mutant PKD1 polypeptides and peptide portions thereof that
are substantially identical to the PKD1 polypeptide SEQ ID NO:2 or
peptide portions thereof, which cause ADPKD symptoms, are
encompassed within the scope of the invention. Such mutant PKD1
polypeptides and peptide portions thereof can include dominant
mutant PKD1 polypeptides, or PKD1 related polypeptides functionally
equivalent to such mutant PKD1 polypeptides. Examples of mutant
PKD1 polypeptide sequences include a polypeptide sequences
substantially identical to SEQ ID NO:2 having one or more amino
acid substitutions such as A88V, W967R, L2696R, R2985G, R3039C,
V3285I, or H3311R, or truncated after amino acid 3000. A peptide
portion of a mutant PKD1 polypeptide can be 3, 6, 9, 12, 20, 50,
100 or more amino acid residues in length, and includes at least
one of the mutations identified above.
[0090] A PKD1 wild type or mutant polypeptide, or peptide portions
thereof, can be purified from natural sources, as discussed below;
can be chemically synthesized; or can be recombinantly expressed.
For example, one skilled in the art can synthesize peptide
fragments corresponding to a mutated portion of the PKD1
polypeptide as set forth in SEQ ID NO:2 (e.g., including residue
3110) and use the synthesized peptide fragment to generate
polyclonal and monoclonal antibodies. Synthetic polypeptides or
peptides can be prepared by chemical synthesis, for example,
solid-phase chemical peptide synthesis methods, which are well
known (see, for example, Merrifield, J. Am. Chem. Soc.,
85:2149-2154, 1963; Stewart and Young, Solid Phase Peptide
Synthesis, 2d ed., Pierce Chemical Co., Rockford Ill., pages
11-12), and have been employed in commercially available laboratory
peptide design and synthesis kits (Cambridge Research
Biochemicals). Such commercially available laboratory kits have
generally utilized the teachings of Geysen et al., Proc. Natl.
Acad. Sci., USA, 81:3998 (1984) and provide for synthesizing
peptides upon the tips of a multitude of rods or pins, each of
which is connected to a single plate. When such a system is
utilized, a plate of rods or pins is inverted and inserted into a
second plate of corresponding wells or reservoirs, which contain
solutions for attaching or anchoring an appropriate amino acid to
the tips of the pins or rods. By repeating such a process step,
i.e., inverting and inserting the tips of the rods or pins into
appropriate solutions, amino acids are built into desired
peptides.
[0091] A number of available FMOC peptide synthesis systems are
available. For example, assembly of a polypeptide or fragment can
be carried out on a solid support using an Applied Biosystems,
Inc., Model 431A automated peptide synthesizer. Such equipment
provides ready access to the peptides of the invention, either by
direct synthesis or by synthesis of a series of fragments that can
be coupled using other known techniques. Accordingly, methods for
the chemical synthesis of polypeptides and peptides are well-known
to those of ordinary skill in the art, e.g., peptides can be
synthesized by solid phase techniques, cleaved from the resin and
purified by preparative high performance liquid chromatography
(see, e.g., Creighton, 1983, Proteins: Structures and Molecular
Principles, W. H. Freeman & Co., N.Y., pp. 50-60). The
composition of the synthetic peptides can be confirmed by amino
acid analysis or sequencing; e.g., using the Edman degradation
procedure (see e.g., Creighton, 1983, supra at pp. 34-49). Thus,
fragments of the PKD1 polypeptide, variant, or mutant can be
chemically synthesized. Peptides can then be used, for example, to
generate antibodies useful in the detection of PKD1 variants and
mutants, as well as the diagnosis of PKD1-associated disorder
(e.g., ADPKD).
[0092] A PKD1 polypeptide or peptide, including variants or mutants
of the invention, can be substantially purified from natural
sources (e.g., purified from cells) using protein separation
techniques, well known in the art. Such methods can separate the
PKD1 polypeptide away from at least about 90% (on a weight basis),
and from at least about 99% of other proteins, glycoproteins, and
other macromolecules normally found in such natural sources. Such
purification techniques can include, but are not limited to
ammonium sulfate precipitation, molecular sieve chromatography,
and/or ion exchange chromatography. Alternatively, or additionally,
the PKD1 polypeptide, variant, or mutant can be purified by
immunoaffinity chromatography using an immunoabsorbent column to
which an antibody is immobilized that is capable of specifically
binding the PKD1 polypeptide, variant, or mutant. Such an antibody
can be monoclonal or polyclonal in origin. For example, an antibody
that specifically binds to a mutant PKD1 polypeptide does not bind
to a wild-type PKD1 polypeptide or peptide thereof. If the PKD1
polypeptide is glycosylated, the glycosylation pattern can be
utilized as part of a purification scheme via, for example, lectin
chromatography.
[0093] The cellular sources from which the PKD1 polypeptide,
variant, or mutants thereof can be purified include, for example,
those cells that are shown by northern and/or western blot analysis
to express a PKD1 polynucleotide, variant, or mutant sequence.
Preferably, such cellular sources are renal cells including, for
example, renal tubular epithelial cells, as well as biliary duct
cells, skeletal muscle cells, lung alveolar epithelial cell,
placental cells, fibroblasts, lymphoblasts, intestinal epithelial
cells, and endothelial cells. Other sources include biological
fluids, fractionated cells such as organelle preparations, or
tissues obtained from a subject. Examples of biological fluids of
use with the invention are blood, serum, plasma, urine, mucous, and
saliva. Tissue or cell samples can also be used with the invention.
The samples can be obtained by many methods such as cellular
aspiration, or by surgical removal of a biopsy sample.
[0094] PKD1 polypeptides, variants, or mutants of the invention can
be secreted out of the cell. Such extracellular forms of the PKD1
polypeptide or mutants thereof can preferably be purified from
whole tissue rather than cells, utilizing any of the techniques
described above. PKD1 expressing cells such as those described
above also can be grown in cell culture, under conditions well
known to those of skill in the art. PKD1 polypeptide or mutants
thereof can then be purified from the cell media using any of the
techniques discussed above.
[0095] A PKD1 polypeptide, variant, or mutant can additionally be
produced by recombinant DNA technology using the PKD1 nucleotide
sequences, variants and mutants described above coupled with
techniques well known in the art. Alternatively, RNA capable of
encoding PKD1 polypeptides, or peptide fragments thereof, can be
chemically synthesized using, for example, automated or
semi-automated synthesizers (see, for example, "Oligonucleotide
Synthesis", 1984, Gait, ed., IRL Press, Oxford, which is
incorporated herein by reference).
[0096] When used as a component in the assay systems described
herein, the mutant PKD1 polypeptide or peptide can be labeled,
either directly or indirectly, to facilitate detection of a complex
formed between the PKD1 polypeptide and an antibody or nucleic acid
sequence, for example. Any of a variety of suitable labeling
systems can be used including, but not limited to, radioisotopes
such as .sup.125I, enzyme labeling systems such as biotin-avidin or
horseradish peroxidase, which generates a detectable colorimetric
signal or light when exposed to substrate, and fluorescent
labels.
[0097] The present invention also provides antibodies that
specifically bind a PKD1 mutant or PKD1 variant, except that, if
desired, an antibody of the invention can exclude an antibody as
described in U.S. Pat. No. 5,891,628, which is incorporated herein
by reference, or an antibody that that specifically binds a PKD1
mutant as described in U.S. Pat. No. 5,891,628. Antibodies that
specifically bind a mutant PKD1 polypeptide are useful as
diagnostic or therapeutic reagents and, therefore, can be used, for
example, in a diagnostic assay for identifying a subject having or
at risk of having ADPKD, and are particularly convenient when
provided as a kit.
[0098] As used herein, the term "specifically binds," when used in
reference to an antibody and an antigen or epitopic portion
thereof, means that the antibody and the antigen (or epitope) have
a dissociation constant of at least about 1.times.10.sup.-7,
generally at least about 1.times.10.sup.-8, usually at least about
1.times.10.sup.-9, and particularly at least about
1.times.10.sup.-1.degree. or less. Methods for identifying and
selecting an antibody having a desired specificity are well known
and routine in the art (see, for example, Harlow and Lane,
"Antibodies: A Laboratory Manual" (Cold Spring Harbor Pub. 1988),
which is incorporated herein by reference.
[0099] Methods for producing antibodies that can specifically bind
one or more PKD1 polypeptide epitopes, particularly epitopes unique
to a mutant PKD1 polypeptide, are disclosed herein or otherwise
well known and routine in the art. Such antibodies can be
polyclonal antibodies or monoclonal antibodies (mAbs), and can be
humanized or chimeric antibodies, single chain antibodies,
anti-idiotypic antibodies, and epitope-binding fragments of any of
the above, including, for example, Fab fragments, F(ab').sub.2
fragments or fragments produced by a Fab expression library. Such
antibodies can be used, for example, in the detection of PKD1
polypeptides, or mutant PKD1 polypeptides, including variant PKD1
polypeptides, which can be in a biological sample, or can be used
for the inhibition of abnormal PKD1 activity. Thus, the antibodies
can be utilized as part of ADPKD treatment methods, as well as in
diagnostic methods, for example, to detect the presence or amount
of a PKD1 polypeptide.
[0100] For the production of antibodies that bind to PKD1,
including a PKD1 variant or PKD1 mutant, various host animals can
be immunized by injection with a PKD1 polypeptide, mutant
polypeptide, variant, or a portion thereof. Such host animals can
include but are not limited to, rabbits, mice, and rats. Various
adjuvants can be used to increase the immunological response,
depending on the host species, including, but not limited to,
Freund's (complete and incomplete), mineral gels such as aluminum
hydroxide, surface active substances such as lysolecithin, pluronic
polyols, polyanions, peptides, oil emulsions, keyhole limpet
hemocyanin, dinitrophenol, and potentially useful human adjuvants
such as BCG (Bacillus Calmette-Guerin) or Corynebacterium
parvum.
[0101] Antibodies that bind to a mutant PKD1 polypeptide, or
peptide portion thereof, of the invention can be prepared using an
intact polypeptide or fragments containing small peptides of
interest as the immunizing antigen. The polypeptide or a peptide
used to immunize an animal can be derived from translated cDNA or
chemical synthesis, and can be conjugated to a carrier protein, if
desired. Such commonly used carriers that can be chemically coupled
to the peptide include keyhole limpet hemocyanin, thyroglobulin,
bovine serum albumin, tetanus toxoid and others as described above
or otherwise known in the art. The coupled polypeptide or peptide
is then used to immunize the animal and antiserum can be collected.
If desired, polyclonal or monoclonal antibodies can be purified,
for example, by binding to and elution from a matrix to which the
polypeptide or a peptide to which the antibodies were raised is
bound. Any of various techniques commonly used in immunology for
purification and/or concentration of polyclonal antibodies, as well
as monoclonal antibodies, can be used (see for example, Coligan, et
al., Unit 9, Current Protocols in Immunology, Wiley Interscience,
1991, which is incorporated herein by reference).
[0102] Anti-idiotype technology can be used to produce monoclonal
antibodies that mimic an epitope. For example, an anti-idiotypic
monoclonal antibody made to a first monoclonal antibody will have a
binding domain in the hypervariable region that is the image of the
epitope bound by the first monoclonal antibody. Antibodies of the
invention include polyclonal antibodies, monoclonal antibodies, and
fragments of polyclonal and monoclonal antibodies that specifically
bind to a mutant PKD1 polypeptide or peptide portion thereof.
[0103] The preparation of polyclonal antibodies is well known to
those skilled in the art (see, for example, Green et al.,
Production of Polyclonal Antisera, in Immunochemical Protocols
(Manson, ed.), pages 1-5 (Humana Press 1992); Coligan et al.,
Production of Polyclonal Antisera in Rabbits, Rats, Mice and
Hamsters, in Current Protocols in Immunology, section 2.4.1 (1992),
which are incorporated herein by reference). The preparation of
monoclonal antibodies likewise is conventional (see, for example,
Kohler and Milstein, Nature, 256:495, 1975, which is incorporated
herein by reference; see, also Coligan et al., supra, sections
2.5.1-2.6.7; and Harlow et al., supra, 1988). Briefly, monoclonal
antibodies can be obtained by injecting mice with a composition
comprising an antigen, verifying the presence of antibody
production by removing a serum sample, removing the spleen to
obtain B lymphocytes, fusing the B lymphocytes with myeloma cells
to produce hybridomas, cloning the hybridomas, selecting positive
clones that produce antibodies to the antigen, and isolating the
antibodies from the hybridoma cultures.
[0104] Monoclonal antibodies can be isolated and purified from
hybridoma cultures by a variety of well-established techniques.
Such isolation techniques include affinity chromatography with
Protein-A Sepharose, size-exclusion chromatography, and
ion-exchange chromatography (see Coligan et al., sections
2.7.1-2.7.12 and sections 2.9.1-2.9.3; Barnes et al., Purification
of Immunoglobulin G (IgG), in Methods in Molecular Biology, Vol.
10, pages 79-104 (Humana Press 1992)). Methods of in vitro and in
vivo multiplication of hybridoma cells expressing monoclonal
antibodies is well-known to those skilled in the art.
Multiplication in vitro can be carried out in suitable culture
media such as Dulbecco's Modified Eagle Medium or RPMI 1640 medium,
optionally replenished by a mammalian serum such as fetal calf
serum or trace elements and growth-sustaining supplements such as
normal mouse peritoneal exudate cells, spleen cells, bone marrow
macrophages. Production in vitro provides relatively pure antibody
preparations and allows scale-up to yield large amounts of the
desired antibodies. Large scale hybridoma cultivation can be
carried out by homogenous suspension culture in an airlift reactor,
in a continuous stirrer reactor, or in immobilized or entrapped
cell culture. Multiplication in vivo can be carried out by
injecting cell clones into mammals histocompatible with the parent
cells, e.g., syngeneic mice, to cause growth of antibody-producing
tumors. Optionally, the animals are primed with a hydrocarbon,
especially oils such as pristane tetramethylpentadecane prior to
injection. After one to three weeks, the desired monoclonal
antibody is recovered from the body fluid of the animal.
[0105] Therapeutic applications for antibodies disclosed herein are
also part of the present invention. For example, antibodies of the
present invention can be derived from subhuman primate antibodies.
General techniques for raising therapeutically useful antibodies in
baboons can be found, for example, in Goldenberg et al.,
International Application Publication No. WO 91/11465, 1991; Losman
et al., Int. J. Cancer, 46:310, 1990, which are incorporated herein
by reference.
[0106] An anti-PKD1 antibody also can be derived from a "humanized"
monoclonal antibody. Humanized monoclonal antibodies are produced
by transferring mouse complementarity determining regions from
heavy and light variable chains of the mouse immunoglobulin into a
human variable domain, and then substituting human residues in the
framework regions of the murine counterparts. The use of antibody
components derived from humanized monoclonal antibodies obviates
potential problems associated with the immunogenicity of murine
constant regions. General techniques for cloning murine
immunoglobulin variable domains are described, for example, by
Orlandi et al., Proc. Natl. Acad. Sci. USA 86:3833, 1989, which is
incorporated herein by reference. Techniques for producing
humanized monoclonal antibodies are described, for example, by
Jones et al., Nature, 321:522, 1986; Riechmann et al., Nature
332:323, 1988; Verhoeyen et al., Science 239:1534, 1988; Carter et
al., Proc. Natl. Acad. Sci. USA, 89:4285, 1992; Sandhu, Crit. Rev.
Biotech. 12:437, 1992; and Singer et al., J. Immunol. 150:2844,
1993, which are incorporated herein by reference.
[0107] Antibodies of the invention also can be derived from human
antibody fragments isolated from a combinatorial immunoglobulin
library (see, for example, Barbas et al., Methods: A Companion to
Methods in Enzymology, Vol. 2, page 119, 1991; Winter et al., Ann.
Rev. Immunol. 12:433, 1994, which are incorporated herein by
reference). Cloning and expression vectors that are useful for
producing a human immunoglobulin phage library can be obtained, for
example, from Stratagene (La Jolla Calif.).
[0108] In addition, antibodies of the present invention can be
derived from a human monoclonal antibody. Such antibodies are
obtained from transgenic mice that have been "engineered" to
produce specific human antibodies in response to antigenic
challenge. In this technique, elements of the human heavy and light
chain loci are introduced into strains of mice derived from
embryonic stem cell lines that contain targeted disruptions of the
endogenous heavy and light chain loci. The transgenic mice can
synthesize human antibodies specific for human antigens, and the
mice can be used to produce human antibody-secreting hybridomas.
Methods for obtaining human antibodies from transgenic mice are
described by Green et al., Nature Genet., 7:13 (1994); Lonberg et
al., Nature, 368:856 (1994); Taylor et al., Int. Immunol., 6:579
(1994), each of which is incorporated herein by reference.
[0109] Antibody fragments of the invention can be prepared by
proteolytic hydrolysis of an antibody or by expression in E. coli
of DNA encoding the fragment. Antibody fragments can be obtained by
pepsin or papain digestion of whole antibodies by conventional
methods. For example, antibody fragments can be produced by
enzymatic cleavage of antibodies with pepsin to provide a 5S
fragment denoted F(ab').sub.2. This fragment can be further cleaved
using a thiol reducing agent, and optionally a blocking group for
the sulfhydryl groups resulting from cleavage of disulfide
linkages, to produce 3.5S Fab' monovalent fragments. Alternatively,
an enzymatic cleavage using pepsin produces two monovalent Fab'
fragments and an Fc fragment directly. These methods are described,
for example, by Goldenberg, U.S. Pat. Nos. 4,036,945 and 4,331,647,
and references contained therein, each of which in incorporated
herein by reference (see, also, Nisonhoff et al., Arch. Biochem.
Biophys. 89:230, 1960; Porter, Biochem. J. 73:119, 1959; Edelman et
al., Meth. Enzymol. 1:422, 1967; and Coligan et al., at sections
2.8.1-2.8.10 and 2.10.1-2.10.4). Other methods of cleaving
antibodies, such as separation of heavy chains to form monovalent
light-heavy chain fragments, further cleavage of fragments, or
other enzymatic, chemical, or genetic techniques can also be used,
provided the fragments bind to the antigen that is recognized by
the intact antibody.
[0110] Fv fragments comprise an association of V.sub.H and V.sub.L
chains, for example, which can be noncovalent (see Inbar et al.,
Proc. Natl. Acad. Sci. USA 69:2659, 1972). The variable chains also
can be linked by an intermolecular disulfide bond, can be
crosslinked by a chemical such as glutaraldehyde (Sandhu, supra,
1992), or F.sub.v fragments comprising V.sub.H and V.sub.L chains
can be connected by a peptide linker. These single chain antigen
binding proteins (sFv) are prepared by constructing a structural
gene comprising DNA sequences encoding the V.sub.H and V.sub.L
domains connected by an oligonucleotide. The structural gene is
inserted into an expression vector, which is subsequently
introduced into a host cell such as E. coli. The recombinant host
cells synthesize a single polypeptide chain with a linker peptide
bridging the two V domains. Methods for producing sFvs are
described, for example, by Whitlow et al., Methods: A Companion to
Meth. Enzymol., 2:97, 1991; Bird et al., Science 242:423, 1988;
Ladner et al., U.S. Pat. No. 4,946,778; Pack et al., BioTechnology
11:1271, 1993; and Sandhu, supra, 1992).
[0111] Another form of an antibody fragment is a peptide coding for
a single complementarity determining region (CDR). CDR peptides
("minimal recognition units") can be obtained by constructing genes
encoding the CDR of an antibody of interest. Such genes are
prepared, for example, by using the polymerase chain reaction to
synthesize the variable region from RNA of antibody-producing cells
(see, for example, Larrick et al., Methods: A Companion to Meth.
Enzymol., 2:106, 1991).
[0112] A variety of methods can be employed utilizing reagents such
as a mutant PKD1 polynucleotide, or oligonucleotide portion thereof
and antibodies directed against a mutant PKD1 polypeptide or
peptide. Specifically, such reagents can be used for the detection
of the presence of PKD1 mutations, e.g., molecules present in
diseased tissue but absent from, or present in greatly reduced
levels compared or relative to the corresponding non-diseased
tissue.
[0113] The methods described herein can be performed, for example,
by utilizing pre-packaged kits, which can be diagnostic kits,
comprising at least one specific oligonucleotide portion of a PKD1
gene or mutant PKD1 polynucleotide, a primer pair, or an anti-PKD1
antibody reagent as disclosed herein, which can be conveniently
used, for example, in a clinical setting to diagnose subjects
exhibiting PKD1 abnormalities or to detect PKD1-associated
disorders, including ADPKD. Any tissue in which a PKD1
polynucleotide is expressed can be utilized in a diagnostic method
of the invention.
[0114] Nucleic acids from a tissue to be analyzed can be isolated
using procedures that are well known in the art, or a diagnostic
procedures can be performed directly on a tissue section (fixed or
frozen), which can be obtained from a subject by biopsy or
resection, without further purification. Oligonucleotide sequences
of the invention can be used as probes or primers for such in situ
procedures (Nuovo, 1992, PCR in situ hybridization: protocols and
applications, Raven Press, N.Y.). For example, oligonucleotide
probes useful in the diagnostic methods of the invention include
nucleotide sequences having at least 10 contiguous nucleotides and
having a sequence substantially identical to a portion of SEQ ID
NO:1, and including nucleotide 474, wherein nucleotide 474 is a T;
nucleotide 487, wherein nucleotide 487 is an A; nucleotide 3110,
wherein nucleotide 3110 is a C; nucleotide 8298, wherein nucleotide
8298 is a G; nucleotide 9164, wherein nucleotide 9164 is a G;
nucleotide 9213, wherein nucleotide 9213 is an A; nucleotide 9326,
wherein nucleotide 9326 is a T; nucleotide 9367, wherein nucleotide
9367 is a T; nucleotide 10064, wherein nucleotide 10064 is an A;
nucleotide 10143, wherein nucleotide 10143 is a G; nucleotide
10234, wherein nucleotide 10234 is a C; nucleotide 10255, wherein
nucleotide 10255 is a T, or a combination thereof. Primers useful
in the present invention include those set forth in SEQ ID NOS:3 to
18 and SEQ ID NOS: 19 to 51 and 61 to 112. Such primers flank and
can be used to amplify sequences containing one or more mutated
nucleotides of a mutant PKD1 polynucleotide.
[0115] PKD1 polynucleotide sequences, either RNA or DNA, can be
used in hybridization or amplification assays of biological samples
to detect abnormalities of PKD1 expression; e.g., Southern or
northern blot analysis, single stranded conformational polymorphism
(SSCP) analysis including in situ hybridization assays, or
polymerase chain reaction analyses, including detecting
abnormalities by a methods such as denaturing high performance
liquid chromatography (DHPLC; also referred to as
temperature-modulated heteroduplex chromatography) or conformation
sensitive gel electrophoresis (CSGE), both of which are readily
adaptable to high throughput analysis (see, for example, Kristensen
et al., BioTechniques 30:318-332, 2001; Leung et al., BioTechniques
30:334-340, 2001, which are incorporated herein by reference). Such
analyses can reveal quantitative abnormalities in the expression
pattern of the PKD1 polynucleotide, and, if the PKD1 mutation is,
for example, an extensive deletion, or the result of a chromosomal
rearrangement, can reveal more qualitative aspects of the PKD1
abnormality.
[0116] Diagnostic methods for detecting a mutant PKD1
polynucleotide can involve, for example, contacting and incubating
nucleic acids derived from a tissue sample being analyzed, with one
or more labeled oligonucleotide probes of the invention or with a
primer or primer pair of the invention, under conditions favorable
for the specific annealing of these reagents to their complementary
sequences within the target molecule. After incubation,
non-annealed oligonucleotides are removed, and hybridization of the
probe or primer, if any, to a nucleic acid from the target tissue
is detected. Using such a detection scheme, the target tissue
nucleic acid can be immobilized, for example, to a solid support
such as a membrane, or a plastic surface such as that on a
microtiter plate or polystyrene beads. In this case, after
incubation, non-annealed, labeled nucleic acid reagents are easily
removed. Detection of the remaining, annealed, labeled nucleic acid
reagents is accomplished using standard techniques well known to
those in the art.
[0117] Oligonucleotide probes or primers of the invention also can
be associated with a solid matrix such as a chip in an array, thus
providing a means for high throughput methods of analysis.
Microfabricated arrays of large numbers of oligonucleotide probes
(DNA chips) are useful for a wide variety of applications.
Accordingly, methods of diagnosing or detecting a PKD1 variant or
mutant can be implemented using a DNA chip for analysis of a PKD1
polynucleotide and detection of mutations therein. A methodology
for large scale analysis on DNA chips is described by Hacia et al.
(Nature Genet. 14:441-447, 1996; U.S. Pat. No. 6,027,880, which are
incorporated herein by reference; see, also, Kristensen et al.,
supra, 2001). As described in Hacia et al., high density arrays of
over 96,000 oligonucleotides, each about 20 nucleotides in length,
are immobilized to a single glass or silicon chip using light
directed chemical synthesis. Contingent on the number and design of
the oligonucleotide probe, potentially every base in a sequence can
be examined for alterations.
[0118] Polynucleotides or oligonucleotides applied to a chip can
contain sequence variations, which can be used to identify
mutations that are not yet known to occur in the population, or
they can only those mutations that are known to occur, including
those disclosed herein (see Example 2). Examples of
oligonucleotides that can be applied to the chip include
oligonucleotides containing at least 10 contiguous nucleotides and
having a sequence substantially identical to a portion of SEQ ID
NO:1, and including nucleotide 474, wherein nucleotide 474 is a T;
nucleotide 487, wherein nucleotide 487 is an A; nucleotide 3110,
wherein nucleotide 3110 is a C; a position corresponding to
nucleotide 3336, wherein nucleotide 3336 is deleted; nucleotide
3707, wherein nucleotide 3707 is an A; nucleotide 4168, wherein
nucleotide 4168 is a T; nucleotide 4885, wherein nucleotide 4885 is
an A; nucleotide 5168, wherein nucleotide 5168 is a T; nucleotide
6058, wherein nucleotide 6058 is a T; nucleotide 6078, wherein
nucleotide 6078 is an A; nucleotide 6089, wherein nucleotide 6089
is a T; nucleotide 6195, wherein nucleotide 6195 is an A;
nucleotide 6326, wherein nucleotide 6326 is a T; a position
corresponding to nucleotides 7205 to 7211, wherein nucleotides 7205
to 7211 are deleted; nucleotide 7376, wherein nucleotide 7376 is a
C; a nucleotide sequence corresponding to nucleotides 7535 to 7536,
wherein a GCG nucleotide sequence is inserted between nucleotides
7535 and 7536; nucleotide 7415, wherein nucleotide 7415 is a T;
nucleotide 7433, wherein nucleotide 7433 is a T; nucleotide 7696,
wherein nucleotide 7696 is a T; nucleotide 7883, wherein nucleotide
7883 is a T; nucleotide 8021, wherein nucleotide 8021 is an A; a
nucleotide sequence corresponding to nucleotide 8159 to 8160,
wherein nucleotides 8159 to 8160 are deleted; nucleotide 8298,
wherein nucleotide 8298 is a G; nucleotide 9164, wherein nucleotide
9164 is a G; nucleotide 9213, wherein nucleotide 9213 is an A;
nucleotide 9326, wherein nucleotide 9326 is a T; nucleotide 9367,
wherein nucleotide 9367 is a T; nucleotide 10064, wherein
nucleotide 10064 is an A; nucleotide 10143, wherein nucleotide
10143 is a G; nucleotide 10234, wherein nucleotide 10234 is a C;
nucleotide 10255, wherein nucleotide 10255 is a T; or a combination
thereof.
[0119] Prior to hybridization with oligonucleotide probes on the
chip, the test sample is isolated, amplified and labeled (e.g.,
fluorescent markers). The test polynucleotide sample is then
hybridized to the immobilized oligonucleotides. The intensity of
sequence-based techniques of the target polynucleotide to the
immobilized probe is quantitated and compared to a reference
sequence. The resulting genetic information can be used in
molecular diagnosis. A common utility of the DNA chip in molecular
diagnosis is screening for known mutations.
[0120] In addition to DNA chip methodology, methods using machinery
adapted to DNA analysis can allow for commercialization of the
disclosed methods of detection of PKD1 mutations and diagnosis of
ADPKD. For example, genotyping by mass spectrometry can be used, or
matrix-assisted laser desorption/ionization-time-of-flight
(MALDI-TOF) mass spectrometry can be used for mass genotyping of
single-base pair and short tandem repeat mutant and variant
sequences. For example, PCR amplification of the region of the
mutation with biotin attached to one of the primers can be
conducted, followed by immobilization of the amplified DNA to
streptavidin beads. Hybridization of a primer adjacent to the
variant or mutant site is performed, then extension with DNA
polymerase past the variant or mutant site in the presence of dNTPs
and ddNTPs is performed. When suitably designed according to the
sequence, this results in the addition of only a few additional
bases (Braun, Little, Koster, 1997). The DNA is then processed to
remove unused nucleotides and salts, and the short primer plus
mutant site is removed by denaturation and transferred to silicon
wafers using a piezoelectric pipette. The mass of the
primer+variant or mutant site is then determined by delayed
extraction MALDI-TOF mass spectrometry. Single base pair and tandem
repeat variations in sequence are easily determined by their mass.
This final step is very rapid, requiring only 5 sec per assay, and
all of these steps can be automated, providing the potential of
performing up to 20,000 genotypings per day. This technology is
rapid, extremely accurate, and adaptable to any variant or
mutation, can identify both single base pair and short tandem
repeat variants, and adding or removing variant or mutant sequences
to be tested can be done in a few seconds at trivial cost.
[0121] Another diagnostic methods for the detection of mutant PKD1
polynucleotides involves amplification, for example, by PCR (see
U.S. Pat. No. 4,683,202), ligase chain reaction (Barany, Proc.
Natl. Acad. Sci. USA 88:189-193, 1991a), self sustained sequence
replication (Guatelli et al., Proc. Natl. Acad. Sci. USA
87:1874-1878, 1990), transcriptional amplification system (Kwoh et
al., Proc. Natl. Acad. Sci. USA 86:1173-1177, 1989), Q-Beta
Replicase (Lizardi et al., Bio/Technology 6:1197, 1988), or any
other RNA amplification method, followed by the detection of the
amplification products. The present invention provides reagents,
methods and compositions that can be used to overcome prior
difficulties with diagnosing ADPKD.
[0122] Using the primer pairs and methods described herein, the
entire replicated segment of the PKD1 gene, including exons 1 and
22, can be amplified from genomic DNA to generate a set of eight
long range amplification products, which range in size from about
0.3 kb to 5.8 kb (Table 1; see, also, FIG. 1). The availability of
widely scattered PKD1-specific primers provides a means to anchor
PKD1-specific amplification, and the ability to use various primer
combinations provides a means to produce longer or shorter
amplification products as desired. For example, the largest PKD1
fragment, which is amplified by primers BPF13 and KG8R25 (see Table
1; SEQ ID NOS: 17 and 18, respectively), can be divided into two
shorter segments by using the PKD1-specific primer, KG85R25 (SEQ ID
NO:18), with forward nested primer F32 (5'-GCCTTGCGCAGCTTGGACT-3;
SEQ ID NO:53), and using BPF13 (SEQ ID NO:17) and a second specific
primer, 31R (5'-ACAGTGTCTTGAGTCCAAGC-3'; SEQ ID NO:54).
[0123] It should be recognized that, while many of the primers
disclosed herein are positioned with intronic sequences of the PKD1
gene, others such as SEQ ID NO:16 are positioned in coding
sequences. As such, a cDNA molecule can obtained from a target RNA
molecule, for example, by reverse transcription of the RNA molecule
using a primer such as SEQ ID NO:16 and an appropriate second
primer positioned 5' or 3' to SEQ ID NO:16. In this embodiment, a
PKD1 RNA can be isolated from any tissue in which wild type PKD1 is
known to be expressed, including, for example, kidney tissue,
nucleated peripheral blood cells, and fibroblasts. A target
sequence within the cDNA is then used as the template for a nucleic
acid amplification reaction, such as a PCR amplification reaction,
or the like. An amplification product can be detected, for example,
using radioactively or fluorescently labeled nucleotides or the
like and an appropriate detection system, or by generating a
sufficient amount of the amplification product such that it can be
visualized by ethidium bromide staining and gel
electrophoresis.
[0124] Genomic DNA from a subject, including from a cell or tissue
sample, can be used as the template for generating a long range
PKD1-specific amplification product. Methods of isolating genomic
DNA are well known and routine (see Sambrook et al., supra, 1989).
Amplification of the genomic PKD1 DNA has advantages over the cDNA
amplification process, including, for example, allowing for
analysis of exons and introns of the PKD1 gene. As such, a target
sequence of interest associated with either an intron or exon
sequence of a PKD1 gene can be amplified and characterized. A
target sequence of interest is any sequence or locus of a PKD1 gene
that contains or is thought to contain a mutation, including those
mutations that correlate to a PKD1-associated disorder or
disease.
[0125] Using primers flanking the target sequence, a sufficient
number of PCR cycles is performed to provide a PKD1-specific
amplification product corresponding to the target sequence. If
desired, additional amplification can be performed, for example, by
performing a nested PCR reaction. Examples of primers useful for
generating a PKD1-specific first amplification product from genomic
DNA include the primer pairs having sequences as exemplified in SEQ
ID NO:3 and 4; SEQ ID NOS:5 and 6; SEQ ID NOS:7 and 8; SEQ ID NOS:9
and 10; SEQ ID NOS:11 and 12; SEQ ID NOS:13 and 14; SEQ ID NOS:15
and 16; and SEQ ID NOS:17 and 18. The PKD1-specific first
amplification product can be further amplified using nested primers
specific for a target sequence, including the primer pairs
exemplified as SEQ ID NOS:19 and 20; SEQ ID NOS:21 and 22; SEQ ID
NOS:23 and 24; SEQ ID NOS:25 and 26; SEQ ID NOS:27 and 28; SEQ ID
NOS:29 and 30; SEQ ID NOS:31 and 32; SEQ ID NOS:33 and 34; SEQ ID
NOS:35 and 36; SEQ ID NOS:37 and 38; SEQ ID NOS:39 and 40; SEQ ID
NOS:41 and 42; SEQ ID NOS:43 and 44; SEQ ID NOS:45 and 46; SEQ ID
NOS:47 and 48; SEQ ID NOS:49 and 50; SEQ ID NOS:51 and 61; and the
primer pairs formed using consecutive primers set forth in Table 2
as SEQ ID NOS:62 to 96, 113, and 97 to 112.
[0126] The amplified target sequences can be examined for changes
(i.e., mutations) with respect to SEQ ID NO:1 using any of various
well known methods as disclosed herein or otherwise known in the
art. For example, the amplification products can simply be
sequenced using routine DNA sequencing methods, particularly where
only one or few amplification products are to be examined. However,
DNA sequencing will be more valuable as a method of detecting
mutations according to a method of the invention as sequencing
technology improves and becomes more adaptable to high throughput
screening assays. In addition, methods that are useful for
detecting the presence of a mutation in a DNA sequence include, for
example, DHPLC (Huber et al., Nucl. Acids Res. 21:1061-10666, 1993;
Liu et al., Nucl. Acids Res. 26:1396-1400, 1998; Choy et al., Ann.
Hum. Genet. 63:383-391, 1999; Ellis et al., Hum. Mutat. 15:556-564,
2000; which are incorporated herein by reference; see, also,
Kristensen et al., supra, 2001); CSGE (Leung et al., supra, 2001);
single-stranded conformation analysis (SSCA; Orita et al., Proc.
Natl. Acad. Sci., USA 86:2766-2770, 1989); denaturing gradient gel
electrophoresis (DGGE; Sheffield et al., Proc. Natl. Acad. Sci.,
USA 86:232-236, 1989); RNase protection assays; allele-specific
oligonucleotides (ASOs; Handelin and Shuber, Current Protocols in
Human Genetics, Suppl. 16 (John Wiley & Sons, Inc. 1998),
9:9.4.1-9.4.8); the use of proteins that recognize nucleotide
mismatches, such as the E. coli mutS protein; and allele-specific
PCR.
[0127] For allele-specific PCR, primers are used that hybridize at
their 3' ends to a particular mutations. Examples of primers that
can be used for allele specific PCR include an oligonucleotide of
at least 10 nucleotide of SEQ ID NO:1 and that has at its 3' end
nucleotide 474, wherein nucleotide 474 is a T; nucleotide 487,
wherein nucleotide 487 is an A; nucleotide 3110, wherein nucleotide
3110 is a C; nucleotide 8298, wherein nucleotide 8298 is a G;
nucleotide 9164, wherein nucleotide 9164 is a G; nucleotide 9213,
wherein nucleotide 9213 is an A; nucleotide 9326, wherein
nucleotide 9326 is a T; nucleotide 9367, wherein nucleotide 9367 is
a T; nucleotide 10064, wherein nucleotide 10064 is an A; nucleotide
10143, wherein nucleotide 10143 is a G; nucleotide 10234, wherein
nucleotide 10234 is a C; or nucleotide 10255, wherein nucleotide
10255 is a T. If the particular mutation is not present, an
amplification product is not observed. Amplification Refractory
Mutation System (ARMS) can also be used (see European Patent
Application Publ. No. 0332435; Newton et al., Nucl. Acids. Res.
17:2503-2516, 1989).
[0128] In the SSCA, DGGE and RNase protection methods, a
distinctive electrophoretic band appears. SSCA detects a band that
migrates differentially because the sequence change causes a
difference in single-strand, intramolecular base pairing. RNase
protection involves cleavage of the mutant polynucleotide into two
or more smaller fragments. DGGE detects differences in migration
rates of mutant sequences compared to wild-type sequences, using a
denaturing gradient gel. In an allele-specific oligonucleotide
assay, an oligonucleotide is designed that detects a specific
sequence, and the assay is performed by detecting the presence or
absence of a hybridization signal. In the mutS assay, the protein
binds only to sequences that contain a nucleotide mismatch in a
heteroduplex between mutant and wild-type sequences.
[0129] Denaturing gradient gel electrophoresis is based on the
melting behavior of the DNA fragments and the use of denaturing
gradient gel electrophoresis as shown by Fischer and Lerman, Proc.
Natl. Acad. Sci. USA 80:1579-83, 1983; Myers et al.; Nucl. Acids
Res. 13:3111-3129, 1985; Lerman et al., in Molecular Biol. of Homo
Sapiens, Cold Spring Harbor Lab. (1986) pp. 285-297. DNA fragments
differing by single base substitutions can be separated from each
other by electrophoresis in polyacrylamide gels containing an
ascending gradient of the DNA denaturants urea and formamide. Two
identical DNA fragments differing by only one single base pair,
will initially move through the polyacrylamide gel at a constant
rate. As they migrate into a critical concentration of denaturant,
specific domains within the fragments melt to produce partially
denatured DNA. Melting of a domain is accompanied by an abrupt
decrease in mobility. The position in the denaturant gradient gel
at which the decrease in mobility is observed corresponds to the
melting temperature of that domain. Since a single base
substitution within the melting domain results in a melting
temperature difference, partial denaturation of the two DNA
fragments will occur at different positions in the gel. DNA
molecules can therefore be separated on the basis of very small
differences in the melting temperature. Additional improvements to
this DGGE have been made as disclosed by Borresen in U.S. Pat. No.
5,190,856. In addition, after a first DGGE analysis, an identified
product can be cloned, purified and analyzed a second time by
DGGE.
[0130] Denaturing high performance liquid chromatography (DHPLC;
Kristensen et al., supra, 2001) and high throughput conformation
sensitive gel electrophoresis (HTCSGD; Leung et al., supra, 2001)
are particularly useful methods for detecting a mutant PKD1
polynucleotide sequence because the methods are readily adaptable
to high throughput analysis. In addition, these methods are
suitable for detecting known mutations as well as identifying
previously unknown mutations. As such, these methods of detection
can be adopted for use in clinical diagnostic settings. DHPLC, for
example, can be used to rapidly screen a large number of samples,
for example, 96 samples prepared using a 96 well microtiter plate
format, to identify those showing a change in the denaturation
properties. Where such a change is identified, confirmation that
the PKD1 polynucleotide in the sample showing the altered
denaturation property is a mutant PKD1 polynucleotide can be
confirmed by DNA sequence analysis, if desired.
[0131] An oligonucleotide probe specific for a mutant PKD1
polynucleotide also can be used to detect a mutant PKD1
polynucleotide in a biological sample, including in a biological
fluid, in cells or tissues obtained from a subject, or in a
cellular fraction such as an organelle preparation. Cellular
sources useful as samples for identifying a mutant PKD1
polynucleotide include, for example, renal cells including renal
tubular epithelial cells, bile duct cells, skeletal muscle cells,
lung alveolar epithelial cells, placental cells, fibroblasts and
lymphocytes. Biological fluids useful as samples for identifying a
mutant PKD1 polynucleotide include, for example, whole blood or
serum or plasma fractions, urine, mucous, and saliva. A biological
sample such as a tissue or cell sample can be obtained by any
method routinely used in a clinical setting, including, for
example, by cellular aspiration, biopsy or other surgical
procedure.
[0132] The oligonucleotide probe can be labeled with a compound
that allows detection of binding to a mutant PKD1 polynucleotide in
the sample. A detectable compound can be, for example, a
radioactive label, which provides a highly sensitive means for
detection, or a non-radioactive label such as a fluorescent,
luminescent, chemiluminescent, or enzymatically detectable label or
the like (see, for example, Matthews et al., Anal. Biochem.
169:1-25, 1988).
[0133] The method of detection can be a direct or indirect method.
An indirect detection process can involve, for example, the use of
an oligonucleotide probe that is labeled with a hapten or ligand
such as digoxigenin or biotin. Following hybridization, the
target-probe duplex is detected by the formation of an antibody or
streptavidin complex, which can further include an enzyme such as
horseradish peroxidase, alkaline phosphatase, or the like. Such
detection systems can be prepared using routine methods, or can be
obtained from a commercial source. For example, the GENIUS
detection system (Boehringer Mannheim) is useful for mutational
analysis of DNA, and provides an indirect method using digoxigenin
as a tag for the oligonucleotide probe and an
anti-digoxigenin-antibody-alkaline phosphatase conjugate as the
reagent for identifying the presence of tagged probe.
[0134] Direct detection methods can utilize, for example,
fluorescent labeled oligonucleotides, lanthanide chelate labeled
oligonucleotides or oligonucleotide-enzyme conjugates. Examples of
fluorescent labels include fluorescein, rhodamine and
phthalocyanine dyes. Examples of lanthanide chelates include
complexes of europium (Eu.sup.3+) or terbium (Tb.sup.3+).
Oligonucleotide-enzyme conjugates are particularly useful for
detecting point mutations when using target-specific
oligonucleotides, as they provide very high sensitivities of
detection. Oligonucleotide-enzyme conjugates can be prepared by a
number of methods (Jablonski et al., Nucl. Acids Res. 14:6115-6128,
1986; Li et al., Nucl. Acids. Res. 15:5275-5287, 1987; Ghosh et
al., Bioconjugate Chem. 1:71-76, 1990). The detection of target
nucleic acids using these conjugates can be carried out by filter
hybridization methods or by bead-based sandwich hybridization
(Ishii et al., Bioconjugate Chem. 4:34-41, 1993).
[0135] Methods for detecting a labeled oligonucleotide probe are
well known in the art and will depend on the particular label. For
radioisotopes, detection is by autoradiography, scintillation
counting or phosphor imaging. For hapten or biotin labels,
detection is with antibody or streptavidin bound to a reporter
enzyme such as horseradish peroxidase or alkaline phosphatase,
which is then detected by enzymatic means. For fluorophor or
lanthanide chelate labels, fluorescent signals can be measured with
spectrofluorimeters, with or without time-resolved mode or using
automated microtiter plate readers. For enzyme labels, detection is
by color or dye deposition, for example, p-nitrophenyl phosphate or
5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium for
alkaline phosphatase, and 3,3'-diaminobenzidine-NiCl.sub.2 for
horseradish peroxidase, fluorescence by 4-methyl umbelliferyl
phosphate for alkaline phosphatase, or chemiluminescence by the
alkaline phosphatase dioxetane substrates LumiPhos 530 (Lumigen
Inc., Detroit Mich.) or AMPPD and CSPD (Tropix, Inc.).
Chemiluminescent detection can be carried out with X-ray or
Polaroid film, or by using single photon counting luminometers,
which also is a useful detection format for alkaline phosphatase
labeled probes.
[0136] Mutational analysis can also be carried out by methods based
on ligation of oligonucleotide sequences that anneal immediately
adjacent to each other on a target DNA or RNA molecule (Wu and
Wallace, Genomics 4:560-569, 1989; Landren et al., Science
241:1077-1080, 1988; Nickerson et al., Proc. Natl. Acad. Sci. USA
87:8923-8927, 1990; Barany, supra, 1991a). Ligase-mediated covalent
attachment occurs only when the oligonucleotides are correctly
base-paired. The ligase chain reaction (LCR) and the
oligonucleotide ligation assay (OLA), which utilize the
thermostable Taq ligase for target amplification, are particularly
useful for interrogating mutation loci. The elevated reaction
temperatures permit the ligation reaction to be conducted with high
stringency (Barany, PCR Methods and Applications 1:5-16, 1991b;
Grossman et al., Nucl. Acids. Res. 22:4527-4534, 1994, which are
incorporated herein by reference).
[0137] Analysis of point mutations in DNA can also be carried out
by using PCR and variations thereof. Mismatches can be detected by
competitive oligonucleotide priming under hybridization conditions
where binding of the perfectly matched primer is favored (Gibbs et
al., Nucl. Acids. Res. 17:2437-2448, 1989). In the amplification
refractory mutation system technique (ARMS), primers can be
designed to have perfect matches or mismatches with target
sequences either internal or at the 3' residue (Newton et al.,
supra, 1989). Under appropriate conditions, only the perfectly
annealed oligonucleotide can function as a primer for the PCR
reaction, thus providing a method of discrimination between normal
and mutant sequences.
[0138] Detection of single base mutations in target nucleic acids
can be conveniently accomplished by differential hybridization
techniques using sequence-specific oligonucleotides (Suggs et al.,
Proc. Natl. Acad. Sci. USA 78:6613-6617, 1981; Conner et al., Proc.
Natl. Acad. Sci. USA 80:278-282, 1983; Saiki et al., Proc. Natl.
Acad. Sci. USA 86:6230-6234, 1989). Mutations can be diagnosed on
the basis of the higher thermal stability of the perfectly matched
probes as compared to the mismatched probes. The hybridization
reactions can be carried out in a filter-based format, in which the
target nucleic acids are immobilized on nitrocellulose or nylon
membranes and probed with oligonucleotide probes. Any of the known
hybridization formats can be used, including Southern blots, slot
blots, reverse dot blots, solution hybridization, solid support
based sandwich hybridization, bead-based, silicon chip-based and
microtiter well-based hybridization formats.
[0139] An alternative strategy involves detection of the mutant
sequences by sandwich hybridization methods. In this strategy, the
mutant and wild type target nucleic acids are separated from
non-homologous DNA/RNA using a common capture oligonucleotide
immobilized on a solid support and detected by specific
oligonucleotide probes tagged with reporter labels. The capture
oligonucleotides can be immobilized on microtiter plate wells or on
beads (Gingeras et al., J. Infect. Dis. 164:1066-1074, 1991; Richen
et al., Proc. Natl. Acad. Sci. USA 88:11241-11245, 1991).
[0140] Another method for analysis of a biological sample for
specific mutations in a PKD1 polynucleotide sequence (e.g., mutant
PKD1 polynucleotides, or oligonucleotide portions thereof) is a
multiplexed primer extension method. In this method primer is
hybridized to a nucleic acid suspected of containing a mutation
such that the primer is hybridized 3' to the suspected mutation.
The primer is extended in the presence of a mixture of one to three
deoxynucleoside triphosphates and one of three chain terminating
deoxynucleoside triphosphates selected such that the wild-type
extension product, the mutant DNA-derived extension product and the
primer each are of different lengths. These steps can be repeated,
such as by PCR or RT-PCR, and the resulting primer extended
products and primer are then separated on the basis of molecular
weight to thereby enable identification of mutant DNA-derived
extension product.
[0141] In one aspect of the invention, the OLA is applied for
quantitative mutational analysis of PKD1 polynucleotide sequences
(Grossman, et al., supra, 1994). In this embodiment of the
invention, a thermostable ligase-catalyzed reaction is used to link
a fluorescently labeled common probe with allele-specific probes.
The latter probes are sequence-coded with non-nucleotide mobility
modifiers that confer unique electrophoretic mobilities to the
ligation products.
[0142] Oligonucleotides specific for wild type or mutant PKD1
sequences can be synthesized with different oligomeric nucleotide
or non-nucleotide modifier tails at their 5' termini. Examples of
nucleotide modifiers are inosine or thymidine residues, whereas
examples of non-nucleotide modifiers include pentaethyleneoxide
(PEO) and hexaethyleneoxide (HEO) monomeric units. The
non-nucleotide modifiers are preferred and most preferably PEO is
used to label the probes. When a DNA template is present, a
thermostable DNA ligase catalyzes the ligation of normal and mutant
probes to a common probe bearing a fluorescent label. The PEO tails
modify the mobilities of the ligation products in electrophoretic
gels. The combination of PEO tails and fluorophor labels (TET and
FAM (5-carboxy-fluorescein derivatives)), HEX and JOE
(6-carboxy-fluorescein derivatives), ROX (6-carboxy-x-rhodamine),
or TAMRA (N, N, N', N'-tetramethyl-6-carboxy-rhodamine;
Perkin-Elmer, ABI Division, Foster City Calif.) allow multiplex
analysis based on size and color by providing unique
electrophoretic signatures to the ligation products. The products
are separated by electrophoresis, and fluorescence intensities
associated with wild type and mutant products are used to
quantitate heteroplasmy. Thus, wild type and mutant, including
variant, sequences are detected and quantitated on the basis of
size and fluorescence intensities of the ligation products. This
method further can be configured for quantitative detection of
multiple PKD1 polynucleotide mutations in a single ligation
reaction.
[0143] Mismatch detection or mutation analysis can also be
performed using mismatch specific DNA intercalating agents. Such
agents intercalate at a site having a mismatch followed by
visualization on a polyacrylamide or agarose gel or by
electrocatalysis. Accordingly, PKD1 polynucleotide sequences can be
contacted with probes specific for a PKD1 mutation or probes that
are wild type for an area having a specific mutation under
conditions such that the PKD1 polynucleotide and probe hybridize.
The hybridized sequences are then contacted with a mismatch
intercalating agent and, for example, separated on a gel.
Visualized bands on the gel correspond to a sequence having a
mismatch. If the probes are wild-type probes mismatches will occur
if the target PKD1 sequence contains a mismatch. If the probes are
specific for a mutated sequence mismatches will be present where
the target PKD1 sequence is wild type, but the hybridized or duplex
sequences will not contain mismatches where the probe sequence
hybridizes to a PKD1 sequence containing the same mutation.
[0144] For quantitative analysis of PKD1 mutations using OLA,
oligonucleotide probes are preferably labeled with fluorophor
labels that provide spectrally distinguishable characteristics. In
one embodiment, oligonucleotides are labeled with 5' oligomeric PEO
tails. Synthesis of such 5' labeled oligonucleotides can be carried
out, for example, using an automated synthesizer using standard
phosphoramidite chemistry. Following cleavage from resin and
deprotection with ammonium hydroxide, the
(PEO).sub.n-oligonucleotides can be purified by reverse phase HPLC.
Oligonucleotides with 3'-FAM or TET dyes (Perkin Elmer) and
5'-phosphates can be synthesized and purified by the procedure of
Grossman et al., supra, 1994. The 5'-PEO-labeled probes can be
synthesized to have 5'-PEO-tails of differing lengths to facilitate
distinguishing the ligated probe products both electrophoretically
by size and by spectral characteristics of the fluorophor
labels.
[0145] The oligonucleotide probes are used for identifying mutant
PKD1 polynucleotides, which can be indicative of a PKD1-associated
disorder such as ADPKD. Preferably, the probes are specific for one
or more PKD1 nucleotide positions of SEQ ID NO:1 selected from
nucleotide 474, wherein nucleotide 474 is a T; nucleotide 487,
wherein nucleotide 487 is an A; nucleotide 3110, wherein nucleotide
3110 is a C; nucleotide 8298, wherein nucleotide 8298 is a G;
nucleotide 9164, wherein nucleotide 9164 is a G; nucleotide 9213,
wherein nucleotide 9213 is an A; nucleotide 9326, wherein
nucleotide 9326 is a T; nucleotide 9367, wherein nucleotide 9367 is
a T; nucleotide 10064, wherein nucleotide 10064 is an A; nucleotide
10143, wherein nucleotide 10143 is a G; nucleotide 10234, wherein
nucleotide 10234 is a C; or nucleotide 10255, wherein nucleotide
10255 is a T. The oligonucleotide probes for the OLA assay are
typically designed to have calculated melting temperatures of about
40.degree. C. to 50.degree. C., generally about 48.degree. C., by
the nearest neighbor method (Breslaur et al., Proc. Natl. Acad.
Sci. USA 83:9373-9377, 1986) so that the ligation reaction can be
performed at a temperature range of about 40.degree. C. to
60.degree. C., typically from about 45.degree. C. to about
55.degree. C. The wild type and mutant, including variant,
oligonucleotide probes can be synthesized with various combinations
of PEO oligomeric tails and fluorescein dyes such as TET and FAM.
These combinations of mobility modifiers and fluorophor labels
furnish electrophoretically unique ligation products that can
enable the monitoring of two or more PKD1 nucleotide sites in a
single ligation reaction.
[0146] In one embodiment, a method of diagnosing a PKD1-associated
disorder in a subject is performed by amplifying a portion of a
PKD1 polynucleotide in a nucleic acid sample from a subject
suspected of having a PKD1-associated disorder with at least a
first primer pair to obtain a first amplification product, wherein
said first primer pair is a primer pair of claim 3; amplifying the
first amplification product with at least a second primer pair to
obtain a nested amplification product, wherein the second primer
pair is suitable for performing nested amplification of the first
amplification product; and determining whether the nested
amplification product has a mutation associated with a
PKD1-associated disorder, wherein the presence of a mutation
associated with a PKD1-associated disorder is indicative of a
PKD1-associated disorder, thereby diagnosing a PKD1-associated
disorder in the subject. The method can be performed using a first
primer pair selected from SEQ ID NOS:3 and 4; SEQ ID NOS:5 and 6;
SEQ ID NOS:7 and 8; SEQ ID NOS:9 and 10; SEQ ID NOS:11 and 12; SEQ
ID NOS:13 and 14; SEQ ID NOS:15 and 16; SEQ ID NOS:17 and 18; and a
combination thereof, and a second primer pair selected from SEQ ID
NOS:19 and 20; SEQ ID NOS:21 and 22; SEQ ID NOS:23 and 24; SEQ ID
NOS:25 and 26; SEQ ID NOS:27 and 28; SEQ ID NOS:29 and 30; SEQ ID
NOS:31 and 32; SEQ ID NOS:33 and 34; SEQ ID NOS:35 and 36; SEQ ID
NOS:37 and 38; SEQ ID NOS:39 and 40; SEQ ID NOS:41 and 42; SEQ ID
NOS:43 and 44; SEQ ID NOS:45 and 46; SEQ ID NOS:47 and 48; SEQ ID
NOS:49 and 50; SEQ ID NOS:51 and 61; SEQ ID NOS:62 and 63; SEQ ID
NOS:64 and 65; SEQ ID NOS:66 and 67; SEQ ID NOS:68 and 69; SEQ ID
NOS:70 and 71; SEQ ID NOS:72 and 73; SEQ ID NOS:74 and 75; SEQ ID
NOS:76 and 77; SEQ ID NOS:78 and 79; SEQ ID NOS:80 and 81; SEQ ID
NOS:82 and 83; SEQ ID NOS:84 and 85; SEQ ID NOS:86 and 87; SEQ ID
NOS:88 and 89; SEQ ID NOS:90 and 91; SEQ ID NOS:92 and 93; SEQ ID
NOS:94 and 95; SEQ ID NOS:96 and 113; SEQ ID NOS:97 and 98; SEQ ID
NOS:99 and 100; SEQ ID NOS:101 and 102; SEQ ID NOS:103 and 104; SEQ
ID NOS: 105 and 106; SEQ ID NOS:107 and 108; SEQ ID NOS:109 and
110; or SEQ ID NOS:111 and 112; and a combination thereof.
[0147] In another embodiment, a method of diagnosing a
PKD1-associated disorder in a subject is performed by amplifying a
portion of PKD1 polynucleotide in a nucleic acid sample from a
subject suspected of having a PKD1-associated disorder with a first
primer pair to obtain a first amplification product; amplifying the
first amplification product using a second primer pair to obtain a
second amplification product; and detecting a mutation in the
second amplification product, wherein the mutation comprises SEQ ID
NO:1 wherein nucleotide 3110 is a C; nucleotide 3336 is deleted;
nucleotide 3707 is an A; nucleotide 5168 is a T; nucleotide 6078 is
an A; nucleotide 6089 is a T; nucleotide 6326 is a T; nucleotides
7205 to 7211 are deleted; nucleotide 7415 is a T; nucleotide 7433
is a T; nucleotide 7883 is a T; or nucleotides 8159 to 8160 are
deleted; nucleotide 8298 is a G; nucleotide 9164 is a G; nucleotide
9213 is an A; or nucleotide 9326 is a T; nucleotide 10064 is an A;
or wherein a GCG nucleotide sequence is inserted between
nucleotides 7535 and 7536; or a combination thereof, thereby
diagnosing a PKD1-associated disorder in the subject.
[0148] The present invention also provides a method of identifying
a subject having or at risk of having a PKD1-associated disorder.
Such a method can be performed, for example, by comprising
contacting nucleic acid molecules in a sample from a subject with
at least one primer pair of the invention under conditions suitable
for amplification of a PKD1 polynucleotide by the primer pair,
thereby generating a PKD1-specific amplification product; and
testing an amplification product for the presence or absence of a
mutation indicative of a PKD1-associated disorder, wherein the
absence of the mutation identifies the subject a not having or at
risk of the having a PKD1-associated disorder, and wherein the
presence of the mutation identifies the subject as having or is at
risk of having a PKD1-associated disorder. The primer pair can be,
for example, selected from SEQ ID NO:3 and 4; SEQ ID NO:5 and 6;
SEQ ID NOS:7 and 8; SEQ ID NOS:9 and 10; SEQ ID NOS:11 and 12; SEQ
ID NOS:13 and 14; SEQ ID NOS:15 and 16; or SEQ ID NOS:17 and 18.
The PKD1-associated disorder can be autosomal dominant polycystic
kidney disease, acquired cystic disease, or any other PKD-1
associated disorder, and the subject can be, for example, a
vertebrate, particularly a human subject.
[0149] Such a method is particularly adaptable to a high throughput
format, and, if desired, can include a step of contacting the
PKD1-specific amplification product with at least a second primer
pair, under conditions suitable for nested amplification of the
PKD1-specific amplification product by a second primer pair,
thereby generating a nested amplification product, then testing the
nested amplification product for the presence or absence of a
mutation indicative of a PKD1-associated disorder. The second
primer pair can be any primer pair suitable for nested
amplification of the PKD1-specific amplification product, for
example, a primer pair selected from SEQ ID NOS:19 and 20; SEQ ID
NOS:21 and 22; SEQ ID NOS:23 and 24; SEQ ID NOS:25 and 26; SEQ ID
NOS:27 and 28; SEQ ID NOS:29 and 30; SEQ ID NOS:31 and 32; SEQ ID
NOS:33 and 34; SEQ ID NOS:35 and 36; SEQ ID NOS:37 and 38; SEQ ID
NOS:39 and 40; SEQ ID NOS:41 and 42; SEQ ID NOS:43 and 44; SEQ ID
NOS:45 and 46; SEQ ID NOS:47 and 48; SEQ ID NOS:49 and 50; SEQ ID
NOS:51 and 61; SEQ ID NOS:62 and 63; SEQ ID NOS:64 and 65; SEQ ID
NOS:66 and 67; SEQ ID NOS:68 and 69; SEQ ID NOS:70 and 71; SEQ ID
NOS:72 and 73; SEQ ID NOS:74 and 75; SEQ ID NOS:76 and 77; SEQ ID
NOS:78 and 79; SEQ ID NOS:80 and 81; SEQ ID NOS:82 and 83; SEQ ID
NOS:84 and 85; SEQ ID NOS:86 and 87; SEQ ID NOS:88 and 89; SEQ ID
NOS:90 and 91; SEQ ID NOS:92 and 93; SEQ ID NOS:94 and 95; SEQ ID
NOS:96 and 113; SEQ ID NOS:97 and 98; SEQ ID NOS:99 and 100; SEQ ID
NOS:101 and 102; SEQ ID NOS:103 and 104; SEQ ID NOS: 105 and 106;
SEQ ID NOS:107 and 108; SEQ ID NOS:109 and 110; or SEQ ID NOS:111
and 112; and a combination thereof.
[0150] Testing an amplification product for the presence or absence
of the mutation can be performed using any of various well known
methods for examining a nucleic acid molecule. For example,
nucleotide sequence of the amplification product can be determined,
and compared with the nucleotide sequence of a corresponding
nucleotide sequence of SEQ ID NO:1. The amplification product also
can be tested by determining the melting temperature of the
amplification product, and comparing the melting temperature to the
melting temperature of a corresponding nucleotide sequence of SEQ
ID NO:1. The melting temperature can be determined, for example,
using denaturing high performance liquid chromatography.
[0151] Where a nested amplification is to be performed, the method
can include a step directed to reducing contamination of the
PKD1-specific amplification product by genomic DNA prior to
contacting the PKD1-specific amplification product with the at
least second set of primer pairs. For example, contamination of the
PKD1-specific amplification product can be reduced by diluting the
PKD1-specific amplification product.
[0152] The mutation indicative of a of PKD1 associated disorder can
be, for example, a nucleotide sequence substantially identical to
SEQ ID NO:1, wherein nucleotide 3110 is a C; nucleotide 8298 is a
G; nucleotide 9164 is a G; nucleotide 9213 is an A; nucleotide 9326
is a T; or nucleotide 10064 is an A; or can be a nucleotide
sequence substantially identical to SEQ ID NO:1, wherein nucleotide
3336 is deleted; nucleotide 3707 is an A; nucleotide 5168 is a T;
nucleotide 6078 is an A; nucleotide 6089 is a T; nucleotide 6326 is
a T; nucleotides 7205 to 7211 are deleted; nucleotide 7415 is a T;
nucleotide 7433 is a T; nucleotide 7883 is a T; or nucleotides 8159
to 8160 are deleted; or wherein a GCG nucleotide sequence is
inserted between nucleotides 7535 and 7536.
[0153] Data that is collected pursuant to a step of detecting the
presence or absence of a mutation indicative of a PKD1-associated
disorder in an amplification product, which can be an amplification
product generated according to a method of the invention,
including, for example, a PKD1-specific amplification product or a
nested amplification product, can be accumulated, and can be
formatted into a form that facilitates determining, for example,
whether a subject is at risk of a PKD1-associated disorder. As
such, the data can be formatted into a report that indicates
whether a subject is at risk of a PKD1-associate disorder. The
report can be in any of various forms, including, for example,
contained in a computer random access or read-only memory, or
stored on a diskette, CD, DVD, magnetic tape; presented on a visual
display such as a computer monitor or other cathode ray tube or
liquid crystal display; or printed on paper. Furthermore, the data,
which can be formatted into a report, can be transmitted to a user
interested in or privy to the information. The data or report can
be transmitted using any convenient medium, for example, via the
internet, by facsimile or by mail, depending on the form of the
data or report.
[0154] Also provided is a method of detecting the presence of a
mutant PKD1 polynucleotide in a sample by contacting a sample
suspected of containing a mutant PKD1 polynucleotide with an
oligonucleotide of the invention under conditions that allow the
oligonucleotide to selectively hybridize with a mutant PKD1
polynucleotide; and detecting selective hybridization of the
oligonucleotide and a mutant PKD1 polynucleotide, thereby detecting
the presence of a mutant PKD1 polynucleotide sequence in the
sample. In another embodiment, a method of detecting the presence
of a mutant PKD1 polypeptide in a sample is provided, for example,
by contacting a sample suspected of containing a mutant PKD1
polypeptide with an antibody of the invention under conditions that
allow the antibody to specifically bind a mutant PKD1 polypeptide;
and detecting specific binding of the antibody and the mutant PKD1
polypeptide in the sample, thereby detecting the presence of a
mutant PKD1 polypeptide in a sample. The mutant PKD1 polypeptide
can have a sequence, for example, substantially as set forth in SEQ
ID NO:2, and having a mutation of A88V, W967R, L2696R, R2985G,
W3001X, R3039C, V3285I, H3311R, or a combination thereof (see,
also, Table 4).
[0155] Antibodies that can specifically bind wild type or mutant
PKD1 polypeptides, or peptide portions thereof, can also be used as
ADPKD diagnostic reagents. Such reagents provide a diagnostic
method that can detect the expression of abnormal PKD1 proteins or
of abnormal levels of PKD1 protein expression, including the
detection of mutant PKD1 polypeptides or aberrant cellular
localization of a PKD1 protein. For example, differences in the
size, electronegativity, or antigenicity of the mutant PKD1 protein
relative to a wild type PKD1 protein can be detected.
[0156] Diagnostic methods for the detection of mutant PKD1
polypeptides or peptide portions thereof can involve, for example,
immunoassays wherein epitopes of a mutant PKD1 polypeptide are
detected by their interaction with an anti-PKD1 specific antibody
(e.g., an anti-mutant PKD1 specific antibody). For example, an
antibody that specifically binds to a mutant PKD1 polypeptide does
not bind to a wild-type PKD1 polypeptide or peptide thereof.
Particular epitopes of PKD1 to which antibodies can be developed
include peptides that are substantially identical to SEQ ID NO:2,
and having at least five amino acids, including amino acid residue
88, wherein residue 88 is a V; residue 967, wherein residue 967 is
an R; residue 2696, wherein residue 2696 is an R; residue 2985,
wherein residue 2985 is a G; residue 3039, wherein residue 3039 is
a C; residue 3285, wherein residue 3285 is an I; or residue 3311,
wherein residue 3311 is an R; or a C-terminal peptide including
amino acid residue 3000, where residue 3001 is absent and the
mutant PKD1 polypeptide is truncated due to the presence of a STOP
codon in the encoding mutant PKD1 polynucleotide.
[0157] Antibodies, or fragments of antibodies, such as those
described, above, are useful in the present invention and can be
used to quantitatively or qualitatively detect the presence of wild
type or mutant PKD1 polypeptides or peptide portions thereof, for
example. This can be accomplished, for example, by
immunofluorescence techniques employing a fluorescently labeled
antibody (see below) coupled with light microscopic, flow
cytometric, or fluorimetric detection.
[0158] The antibodies (or fragments thereof) useful in the present
invention can, additionally, be employed histologically, as in
immunofluorescence or immunoelectron microscopy, for in situ
detection of PKD1 polypeptide, peptides, variants or mutants
thereof. Detection can be accomplished by removing a histological
specimen from a subject, and applying thereto a labeled antibody of
the present invention. The histological sample can be taken from a
tissue suspected of exhibiting ADPKD. The antibody (or fragment) is
preferably applied by overlaying the labeled antibody (or fragment)
onto a biological sample. Through the use of such a procedure, it
is possible to determine not only the presence of PKD1
polypeptides, but also their distribution in the examined tissue.
Using the present invention, those of ordinary skill will readily
perceive that any of a wide variety of histological methods (such
as staining procedures) can be modified in order to achieve such in
situ detection.
[0159] Immunoassays for wild type or mutant PKD1 polypeptide or
peptide portions thereof typically comprise incubating a biological
sample, such as a biological fluid, a tissue extract, freshly
harvested cells, or cells that have been incubated in tissue
culture, in the presence of a detectably labeled antibody capable
of identifying a PKD1 polypeptide, mutant PKD1 polypeptide and
peptide portions thereof, and detecting the bound antibody by any
of a number of techniques well-known in the art. The biological
sample can be brought in contact with and immobilized onto a solid
phase support or carrier such as nitrocellulose, or other solid
support that is capable of immobilizing cells, cell particles or
soluble proteins. The support can then be washed with suitable
buffers followed by treatment with the detectably labeled mutant
PKD1 specific antibody, preferably an antibody that recognizes a
developed include peptides that are substantially identical to SEQ
ID NO:2, and having at least five amino acids, including amino acid
residue 88, wherein residue 88 is a V; residue 967, wherein residue
967 is an R; residue 2696, wherein residue 2696 is an R; residue
2985, wherein residue 2985 is a G; residue 3039, wherein residue
3039 is a C; residue 3285, wherein residue 3285 is an I; or residue
3311, wherein residue 3311 is an R; or a C-terminal peptide
including amino acid residue 3000, where residue 3001 is absent and
the mutant PKD1 polypeptide is truncated due to the presence of a
STOP codon in the encoding mutant PKD1 polynucleotide (see, also,
Table 4). The solid phase support can then be washed with the
buffer a second time to remove unbound antibody, and the amount of
bound label on solid support can be detected by conventional means
specific for the label.
[0160] A "solid phase support" or "carrier" can be any support
capable of binding an antigen or an antibody. Well-known supports
or carriers include glass, polystyrene, polypropylene,
polyethylene, dextran, nylon, amylases, natural and modified
celluloses, polyacrylamides, and magnetite. The nature of the
carrier can be either soluble to some extent or insoluble for the
purposes of the present invention. The support material can have
virtually any possible structural configuration so long as the
coupled molecule is capable of binding to an antigen or antibody.
Thus, the support configuration can be spherical, as in a bead, or
cylindrical, as in the inside surface of a test tube, or the
external surface of a rod. Alternatively, the surface can be flat
such as a sheet, test strip, or the like. Those skilled in the art
will know many other suitable carriers for binding antibody or
antigen, or will be able to ascertain the same by use of routine
experimentation.
[0161] The binding activity of a given lot of an anti-mutant PKD1
antibody can be determined according to well known methods. Those
skilled in the art will be able to determine operative and optimal
assay conditions for each determination by employing routine
experimentation. One of the ways in which the mutant PKD1-specific
antibody can be detectably labeled is by linking the antibody to an
enzyme and use the enzyme labeled antibody in an enzyme immunoassay
(EIA; Voller, "The Enzyme Linked Immunosorbent Assay (ELISA),
Diagnostic Horizons 2:1-7, 1978; Microbiological Associates
Quarterly Publication, Walkersville, Md.); Voller et al., J. Clin.
Pathol. 31:507-520, 1978; Butler, Meth. Enzymol. 73:482-523, 1981;
Maggio (ed.), "Enzyme Immunoassay", CRC Press, Boca Raton Fla.,
1980; Ishikawa et al., (eds.), "Enzyme Immunoassay", Kgaku Shoin,
Tokyo, 1981). The enzyme that is bound to the antibody will react
with an appropriate substrate, preferably a chromogenic substrate,
in such a manner as to produce a chemical moiety that can be
detected, for example, by spectrophotometric, fluorimetric or by
visual means.
[0162] Enzymes that can be used to detectably label the antibody
include, but are not limited to, malate dehydrogenase,
staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol
dehydrogenase, I-glycerophosphate, dehydrogenase, triose phosphate
isomerase, horseradish peroxidase, alkaline phosphatase,
asparaginase, glucose oxidase, beta-galactosidase, ribonuclease,
urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase
and acetylcholinesterase. The detection can be accomplished by
colorimetric methods that employ a chromogenic substrate for the
enzyme. Detection can also be accomplished by visual comparison of
the extent of enzymatic reaction of a substrate in comparison with
similarly prepared standards. In addition, detection can be
accomplished using any of a variety of other immunoassays,
including, for example, by radioactively labeling the antibodies or
antibody fragments and detecting PKD1 wild type or mutant peptides
using a radioimmunoassay (RIA; see, for example, Weintraub,
Principles of Radioimmunoassays, Seventh Training Course on
Radioligand Assay Techniques, The Endocrine Society, March, 1986,
which is incorporated herein by reference). The radioactive isotope
can be detected by such means as the use of a gamma counter or a
scintillation counter or by autoradiography.
[0163] The antibody also can be labeled with a fluorescent
compound. When the fluorescently labeled antibody is exposed to
light of the proper wave length, its presence can then be detected
due to fluorescence. Among the most commonly used fluorescent
labeling compounds are fluorescein isothiocyanate, rhodamine,
phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde and
fluorescamine. The antibody can also be detectably labeled using
fluorescence emitting metals such as .sup.152Eu, or others of the
lanthanide series. These metals can be attached to the antibody
using such metal chelating groups as diethylenetriaminepentacetic
acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).
[0164] The antibody also can be detectably labeled by coupling it
to a chemiluminescent compound. The presence of the
chemiluminescent-tagged antibody is then determined by detecting
the presence of luminescence that arises during the course of a
chemical reaction. Examples of particularly useful chemiluminescent
labeling compounds are luminol, isoluminol, theromatic acridinium
ester, imidazole, acridinium salt and oxalate ester. Likewise, a
bioluminescent compound can be used to label the antibody of the
present invention. Bioluminescence is a type of chemiluminescence
found in biological systems in, which a catalytic protein increases
the efficiency of the chemiluminescent reaction. The presence of a
bioluminescent protein is determined by detecting the presence of
luminescence. Important bioluminescent compounds for purposes of
labeling are luciferin, luciferase and aequorin.
[0165] In vitro systems can be designed to identify compounds
capable of binding a mutant PKD1 polynucleotide of the invention
(e.g., a polynucleotide having a sequence substantially identical
to SEQ ID NO:1 and having a mutation such as C474T; G487A; T3110C;
T8298G; A9164G; G9213A; C9326T; C9367T; G10064A; A10143G; T10234C;
or G10255T). Such compounds can include, but are not limited to,
peptides made of D- and/or L-configuration amino acids in, for
example, the form of random peptide libraries (see, e.g., Lam et
al., Nature 354:82-84, 1981), phosphopeptides in, for example, the
form of random or partially degenerate, directed phosphopeptide
libraries (see, e.g., Songyang et al., Cell 72:767-778, 1993),
antibodies, and small or large organic or inorganic molecules.
Compounds identified can be useful, for example, in modulating the
activity of PKD1 proteins, variants or mutants. For example, mutant
PKD1 polypeptides of the invention can be useful in elaborating the
biological function of the PKD1 protein. Such mutants can be
utilized in screens for identifying compounds that disrupt normal
PKD1 interactions, or can in themselves disrupt such
interactions.
[0166] The principle of the assays used to identify compounds that
bind to a mutant PKD1 protein involves preparing a reaction mixture
of the PKD1 protein, which can be a mutant, including a variant,
and the test compound under conditions and for a time sufficient to
allow the two components to interact, then isolating the
interaction product (complex) or detecting the complex in the
reaction mixture. Such assays can be conducted in a heterogeneous
or homogeneous format. Heterogeneous assays involve anchoring PKD1
or the test substance onto a solid phase and detecting PKD1 test
substance complexes anchored on the solid phase at the end of the
reaction. In homogeneous assays, the entire reaction is carried out
in a liquid phase. In either approach, the order of addition of
reactants can be varied to obtain different information about the
compounds being tested.
[0167] In addition, methods suitable for detecting protein-protein
interactions can be employed for identifying novel PKD1 cellular or
extracellular protein interactions based upon the mutant or variant
PKD1 polypeptides of the invention. For example, some traditional
methods that can be employed are co-immunoprecipitation,
crosslinking and copurification through gradients or
chromatographic columns. Additionally, methods that result in the
simultaneous identification of the genes coding for the protein
interacting with a target protein can be employed. These methods
include, for example, probing expression libraries with labeled
target protein, using this protein in a manner similar to antibody
probing of .SIGMA.gt libraries. One such method for detecting
protein interactions in vivo is the yeast two hybrid system. One
version of this system has been described (Chien et al., Proc.
Natl. Acad. Sci. USA 88:9578-9582, 1991) and can be performed using
commercially available reagents (Clontech; Palo Alto Calif.).
[0168] A PKD1 polypeptide (e.g., a variant or mutant) of the
invention can interact with one or more cellular or extracellular
proteins in vivo. Such cellular proteins are referred to herein as
"binding partners". Compounds that disrupt such interactions can be
useful in regulating the activity of a PKD1 polypeptide, especially
mutant PKD1 polypeptides. Such compounds include, for example,
molecules such as antibodies, peptides, peptidomimetics and the
like.
[0169] In instances whereby ADPKD symptoms are associated with a
mutation within the PKD1 polynucleotide (e.g., SEQ ID NO:1 having a
mutation at T3110C; T8298G; A9164G; G9213A; C9326T; G10064A or the
like; see Example 2), which produces PKD1 polypeptides having
aberrant activity, compounds identified that disrupt such activity
can therefore inhibit the aberrant PKD1 activity and reduce or
treat ADPKD1-associated symptoms or ADPKD disease, respectively
(see Table 4). For example, compounds can be identified that
disrupt the interaction of mutant PKD1 polypeptides with cellular
or extracellular proteins, for example, the PKD2 gene product, but
do not substantially effect the interactions of the normal PKD1
protein. Such compounds can be identified by comparing the
effectiveness of a compound to disrupt interactions in an assay
containing normal PKD1 protein to that of an assay containing
mutant PKD1 polypeptide, for example, a two hybrid assay.
[0170] The basic principle of the assay systems used to identify
compounds that interfere with the interaction between the PKD1
protein, preferably a mutant PKD1 protein, and its cellular or
extracellular protein binding partner or partners involves
preparing a reaction mixture containing the PKD1 protein and the
binding partner under conditions and for a time sufficient to allow
the two proteins to interact or bind, thus forming a complex. In
order to test a compound for inhibitory activity, reactions are
conducted in the presence or absence of the test compound, i.e.,
the test compound can be initially included in the reaction
mixture, or added at a time subsequent to the addition of PKD1 and
its cellular or extracellular binding partner; controls are
incubated without the test compound or with a placebo. The
formation of any complexes between the PKD1 protein and the
cellular or extracellular binding partner is then detected. The
formation of a complex or interaction in the control reaction, but
not in the reaction mixture containing the test compound indicates
that the compound interferes with the interaction of the PKD1
protein and the binding partner. As noted above, complex formation
or component interaction within reaction mixtures containing the
test compound and normal PKD1 protein can also be compared to
complex formation or component interaction within reaction mixtures
containing the test compound and mutant PKD1 protein. This
comparison can be important in those cases wherein it is desirable
to identify compounds that disrupt interactions of mutant but not
normal PKD1 proteins.
[0171] Any of the binding compounds, including but not limited to,
compounds such as those identified in the foregoing assay systems
can be tested for anti-ADPKD activity. ADPKD, an autosomal dominant
disorder, can involve underexpression of a wild-type PKD1 allele,
or expression of a PKD1 polypeptide that exhibits little or no PKD1
activity. In such an instance, even though the PKD1 polypeptide is
present, the overall level of normal PKD1 polypeptide present is
insufficient and leads to ADPKD symptoms. As such increase in the
level of expression of the normal PKD1 polypeptide, to levels
wherein ADPKD symptoms are ameliorated would be useful.
Additionally, the term can refer to an increase in the level of
normal PKD1 activity in the cell, to levels wherein ADPKD symptoms
are ameliorated.
[0172] The identified compounds that inhibit PKD1 expression,
synthesis and/or activity can be administered to a patient at
therapeutically effective doses to treat polycystic kidney disease.
A therapeutically effective dose refers to that amount of the
compound sufficient to result in amelioration of symptoms of
polycystic kidney disease. Toxicity and therapeutic efficacy of
such compounds can be determined by standard pharmaceutical
procedures in cell cultures or experimental animals, e.g., for
determining the LD.sub.50 (the dose lethal to 50% of the
population) and the ED.sub.50 (the dose therapeutically effective
in 50% of the population). The dose ratio between toxic and
therapeutic effects is the therapeutic index and it can be
expressed as the ratio LD.sub.50/ED.sub.50. Compounds that exhibit
large therapeutic indices are preferred. While compounds that
exhibit toxic side effects can be used, care should be taken to
design a delivery system that targets such compounds to the site of
affected tissue in order to minimize potential damage to uninfected
cells and, thereby, reduce side effects.
[0173] The data obtained from the cell culture assays and animal
studies can be used in formulating a range of dosage for use in
humans. The dosage of such compounds lies preferably within a range
of circulating concentrations that include the ED.sub.50 with
little or no toxicity. The dosage can vary within this range
depending upon the dosage form employed and the route of
administration utilized. For any compound used in the method of the
invention, the therapeutically effective dose can be estimated
initially from cell culture assays. A dose can be formulated in
animal models to achieve a circulating plasma concentration range
that includes the IC.sub.50 (i.e., the concentration of the test
compound that achieves a half-maximal inhibition of symptoms) as
determined in cell culture. Such information can be used to more
accurately determine useful doses in humans. Levels in plasma can
be measured, for example, by high performance liquid
chromatography. Additional factors that can be utilized to optimize
dosage can include, for example, such factors as the severity of
the ADPKD symptoms as well as the age, weight and possible
additional disorders that the patient can also exhibit. Those
skilled in the art will be able to determine the appropriate dose
based on the above factors.
[0174] Pharmaceutical compositions for use in accordance with the
present invention can be formulated in conventional manner using
one or more physiologically acceptable carriers or excipients.
Thus, the compounds and their physiologically acceptable salts and
solvates can be formulated for administration by inhalation (either
through the mouth or the nose) or oral, buccal, parenteral or
rectal administration.
[0175] For oral administration, the pharmaceutical compositions can
take the form of, for example, tablets or capsules prepared by
conventional means with pharmaceutically acceptable excipients such
as binding agents (e.g., pregelatinised maize starch,
polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers
(e.g., lactose, microcrystalline cellulose or calcium hydrogen
phosphate); lubricants (e.g., magnesium stearate, talc or silica);
disintegrants (e.g., potato starch or sodium starch glycollate); or
wetting agents (e.g., sodium lauryl sulphate). The tablets can be
coated by methods well known in the art. Liquid preparations for
oral administration can take the form of, for example, solutions,
syrups or suspensions, or they can be presented as a dry product
for constitution with water or other suitable vehicle before use.
Such liquid preparations can be prepared by conventional means with
pharmaceutically acceptable additives such as suspending agents
(e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible
fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous
vehicles (e.g., almond oil, oily esters, ethyl alcohol or
fractionated vegetable oils); and preservatives (e.g., methyl or
propyl-p-hydroxybenzoates or sorbic acid). The preparations can
also contain buffer salts, flavoring, coloring and sweetening
agents as appropriate.
[0176] Preparations for oral administration can be suitably
formulated to give controlled release of the active compound. For
buccal administration the compositions can take the form of tablets
or lozenges formulated in conventional manner.
[0177] For administration by inhalation, the compounds for use
according to the present invention are conveniently delivered in
the form of an aerosol spray presentation from pressurized packs or
a nebuliser, with the use of a suitable propellant such as
dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In
the case of a pressurized aerosol the dosage unit can be determined
by providing a valve to deliver a metered amount. Capsules and
cartridges of e.g., gelatin, for use in an inhaler can be
formulated containing a powder mix of the compound and a suitable
powder base such as lactose or starch.
[0178] The compounds can be formulated for parenteral
administration by injection, e.g., by bolus injection or continuous
infusion. Formulations for injection can be presented in unit
dosage form, e.g., in ampoules or in multi-dose containers, with an
added preservative. The compositions can take such forms as
suspensions, solutions or emulsions in oily or aqueous vehicles,
and can contain formulatory agents such as suspending, stabilizing
and/or dispersing agents. Alternatively, the active ingredient can
be in powder form for constitution with a suitable vehicle, e.g.,
sterile pyrogen-free water, before use. The compounds can also be
formulated in rectal compositions such as suppositories or
retention enemas, e.g., containing conventional suppository bases
such as cocoa butter or other glycerides.
[0179] In addition to the formulations described previously, the
compounds can also be formulated as a depot preparation. Such long
acting formulations can be administered by implantation (for
example subcutaneously or intramuscularly) or by intramuscular
injection. Thus, for example, the compounds can be formulated with
suitable polymeric or hydrophobic materials (for example as an
emulsion in an acceptable oil) or ion exchange resins, or as
sparingly soluble derivatives, for example, as a sparingly soluble
salt.
[0180] The compositions can, if desired, be presented in a pack or
dispenser device that can contain one or more unit dosage forms
containing the active ingredient. The pack can for example comprise
metal or plastic foil, such as a blister pack. The pack or
dispenser device can be accompanied by instructions for
administration.
[0181] Alternatively, ADPKD can be caused by the production of an
aberrant mutant form of the PKD1 protein, that either interferes
with the normal allele product or introduces a novel function into
the cell, which then leads to the mutant phenotype. For example, a
mutant PKD1 protein can compete with the wild type protein for the
binding of a substance required to relay a signal inside or outside
of a cell.
[0182] Cell based and animal model based assays for the
identification of compounds exhibiting anti-ADPKD activity are also
encompassed within the present invention. Cells that contain and
express mutant PKD1 polynucleotide sequences (e.g., a sequence
substantially identical to the sequence as set forth in SEQ ID NO:1
and having one or more mutations of a C474T; G487A; T3110C; T8298G;
A9164G; G9213A; C9326T; C9367T; G10064A; A10143G; T10234C; G10255T
or the like; see Example 2), which encode a mutant PKD1
polypeptide, and thus exhibit cellular phenotypes associated with
ADPKD, can be utilized to identify compounds that possess
anti-ADPKD activity. Such cells can include cell lines consisting
of naturally occurring or engineered cells that express mutant or
express both normal and mutant PKD1 polypeptides. Such cells
include, but are not limited to renal epithelial cells, including
primary and immortalized human renal tubular cells, MDCK cells,
LLPCK1 cells, and human renal carcinoma cells. Methods of
transforming cell with PKD1 polynucleotide sequences encoding
wild-type or mutant proteins are described above.
[0183] Cells that exhibit ADPKD-like cellular phenotypes, can be
exposed to a compound suspected of exhibiting anti-ADPKD activity
at a sufficient concentration and for a time sufficient to elicit
an anti-ADPKD1 activity in the exposed cells. After exposure, the
cells are examined to determine whether one or more of the
ADPKD-like cellular phenotypes has been altered to resemble a more
wild type, non-ADPKD phenotype.
[0184] Among the cellular phenotypes that can be followed in the
above assays are differences in the apical/basolateral distribution
of membrane proteins. For example, normal (i.e., non-ADPKD) renal
tubular cells in situ and in culture under defined conditions have
a characteristic pattern of apical/basolateral distribution of cell
surface markers. ADPKD renal cells, by contrast, exhibit a
distribution pattern that reflects a partially reversed
apical/basolateral polarity relative to the normal distribution.
For example, sodium-potassium ATPase generally is found on the
basolateral membranes of renal epithelial cells, but also can be
found on the apical surface of ADPKD epithelial cells, both in
cystic epithelia in vivo and in ADPKD cells in culture (Wilson et
al., Am. J. Physiol. 260:F420-F430, 1991). Another marker that
exhibits an alteration in polarity in normal versus ADPKD affected
cells is the EGF receptor, which is normally located basolaterally,
but in ADPKD cells is mislocated to the apical surface. Such a
apical/basolateral marker distribution phenotype can be followed,
for example, by standard immunohistology techniques using
antibodies specific to a markers of interest.
[0185] Assays for the function of PKD1 also can include a measure
of the rate of cell growth or apoptosis, since dysregulation of
epithelial cell growth can be a key step in cyst formation. The
cysts are fluid filled structures lined by epithelial cells that
are both hyper-proliferative and hyper-apoptotic (Evan et al.,
Kidney International 16:743-750, 1979; Kovacs and Gomba, Kidney
Blood Press. Res. 21:325-328, 1998; Lanoix et al., Oncogene 13:
1153-1160, 1996; Woo, New Engl. J. Med. 333:18-25, 1995, each of
which is incorporated herein by reference). The cystic epithelium
has a high mitotic rate in vivo as measured by PCNA staining
(Nadasdy et al., J. Am. Soc. Nephrol. 5:1462-1468, 1995, which is
incorporated herein by reference), and increased levels of
expression of other markers of proliferation (Klingel et al., Amer.
J. Kidney Dis. 19:22-30, 1992, which is incorporated herein by
reference). In addition, cultured cells from ADPKD cystic kidneys
have increased growth rates in vitro (Wilson et al., Kidney Int.
30:371-380, 1986; Wilson, Amer. J. Kidney Dis. 17:634-637, 1991,
each of which is incorporated herein by reference).
[0186] Further, in studies of rodent models of polycystic kidney
disease, the epithelial cells that line cysts of animals with
naturally occurring forms of PKD showed abnormalities similar to
those reported in human ADPKD (Harding et al., 1992; Ramasubbu et
al., J. Am. Soc. Nephrol. 9:937-945, 1998; Rankin et al., J. Cell
Physiol. 152:578-586, 1992; Rankin et al., In Vitro Cell Devel.
Biol. Anim. 32:100-106, 1996, each of which is incorporated herein
by reference). Moreover, mice that have transgenic overexpression
of either c-myc or SV40-large T antigen developed PKD (Kelley et
al. J. Am. Soc. Nephrol. 2:84-97, 1991; Trudel et al., Kidney Int.
39:665-671 1991, each of which is incorporated herein by
reference). Also, expression of recombinant full length PKD1 in
epithelial cells reduced their rate of growth and induced
resistance to apoptosis when challenged with stimuli such as serum
starvation or exposure to UV light, which are known to stimulate
apoptosis (Boletta et al., Mol. Cell 6:1267-1273, 2000, which is
incorporated herein by reference). As such, biochemical pathways
that are activated by PKD1 expression, including, for example,
JAK2, STAT1/3, PI3 kinase, p21, and AKT, can provide surrogate
markers for PKD1 activity.
[0187] The propensity of an epithelial cell to form tubules
provides still another assay for the function of PKD1. In vivo, PKD
is characterized by cystic transformation of renal tubules and
pancreatic and biliary ductules. In vitro, expression of full
length PKD1 induces spontaneous tubulogenesis in MDCK cells
(Boletta et al., supra, 2000). In this model system, control MDCK
cells, which did not express recombinant wild type full length
PKD1, formed cystic structures unless treated with hepatocyte
growth factor or with fibroblast conditioned medium when cultured
suspended in collagen. In contrast, MDCK cells that expressed the
full length wild type recombinant form of PKD1 spontaneously formed
tubules in the absence of exogenous factors when cultured in this
manner. As such, this model system can be used to identify ligands
that bind to and activate the PKD1 protein, to determine pathways
that are targeted for activation by therapeutic agents, and as an
assay system to evaluate the effect of sequence variants on PKD1
function.
[0188] Additionally, assays for the function of a PKD1 polypeptide
can, for example, include a measure of extracellular matrix (ECM)
components, such as proteoglycans, laminin, fibronectin and the
like, in that studies in both ADPKD and in rat models of acquired
cystic disease (Carone et al., Kidney International 35:1034-1040,
1989) have shown alterations in such components. Thus, any compound
that serves to create an extracellular matrix environment that more
fully mimics the normal ECM should be considered as a candidate for
testing for an ability to ameliorate ADPKD symptoms.
[0189] In addition, it is contemplated that the present invention
can be used to measure the ability of a compound, such as those
identified in the foregoing binding assays, to prevent or inhibit
disease in animal models for ADPKD. Several naturally-occurring
mutations for renal cystic disease have been found in animals, and
are accepted in the art as models of ADPKD and provide test systems
for assaying the effects of compounds that interact with PKD1
proteins. Of these models, the Han:SPRD rat model, provides an
autosomal dominant model system (see, for example,
Kaspareit-Rittinghausen et al., Vet. Path. 26:195, 1989), and
several recessive models also are available (Reeders, Nature
Genetics 1:235, 1992). In addition, knock-out mice, in which the
PKD1 or PKD2 gene has been disrupted, are available and provide a
relevant model system for genetic forms of ADPKD. As such, the PKD1
and PKD2 knock-out mice can be useful for confirming the
effectiveness in vivo of compounds that interact with PKD1 proteins
in vitro (see, for example, Wu et al., Nat. Genet. 24:75-78, 2000;
Kim et al., Proc. Natl. Acad. Sci., USA 97:1731-1736, 2000; Lu et
al., Nat. Genet. 21:160-161, 1999; Wu et al., Cell 93:177-188,
1998; Lu et al., Nat. Genet. 17:179-181, 1997, each of which is
incorporated herein by reference).
[0190] Animal models exhibiting ADPKD-like symptoms associated with
one or more of the mutant PKD1 polynucleotide sequences as
disclosed herein can also be engineered by utilizing the PKD1
polynucleotide sequences such in conjunction with well known
methods for producing transgenic animals. Animals of any species,
including, but not limited to, mice, rats, rabbits, guinea pigs,
pigs, mini-pigs, goats, and non-human primates, e.g., baboons,
squirrels, monkeys, and chimpanzees can be used to generate such
ADPKD animal models or transgenic animals. In instances where the
PKD1 mutation leading to ADPKD symptoms causes a drop in the level
of PKD1 protein or causes an ineffective PKD1 protein to be made
(e.g., the PKD1 mutation is a dominant loss-of-function mutation,
such as a W3001X, i.e., truncated after amino acid residue 3000, or
a T3110C mutation; see, also, Table 4) various strategies can be
utilized to generate animal models exhibiting ADPKD-like
symptoms.
[0191] The present invention also provides transgenic non-human
organisms, including invertebrates, vertebrates and mammals. For
purposes of the subject invention, these animals are referred to as
"transgenic" when such animal has had a heterologous DNA sequence,
or one or more additional DNA sequences normally endogenous to the
animal (collectively referred to herein as "transgenes")
chromosomally integrated into the germ cells of the animal. The
transgenic animal (including its progeny) will also have the
transgene integrated into the chromosomes of somatic cells.
[0192] Various methods to make the transgenic animals of the
subject invention can be employed. Generally speaking, three such
methods can be employed. In one such method, an embryo at the
pronuclear stage (a "one cell embryo") is harvested from a female
and the transgene is microinjected into the embryo, in which case
the transgene will be chromosomally integrated into both the germ
cells and somatic cells of the resulting mature animal. In another
such method, embryonic stem cells are isolated and the transgene
incorporated therein by electroporation, plasmid transfection or
microinjection, followed by reintroduction of the stem cells into
the embryo where they colonize and contribute to the germ line.
Methods for microinjection of mammalian species is described in
U.S. Pat. No. 4,873,191.
[0193] In yet another such method, embryonic cells are infected
with a retrovirus containing the transgene whereby the germ cells
of the embryo have the transgene chromosomally integrated therein.
When the animals to be made transgenic are avian, because avian
fertilized ova generally go through cell division for the first
twenty hours in the oviduct, microinjection into the pronucleus of
the fertilized egg is problematic due to the inaccessibility of the
pronucleus. Therefore, of the methods to make transgenic animals
described generally above, retrovirus infection is preferred for
avian species, for example as described in U.S. Pat. No. 5,162,215.
If microinjection is to be used with avian species, however, the
method of Love et al., (Biotechnology, 12, Jan. 1994) can be
utilized whereby the embryo is obtained from a sacrificed hen
approximately two and one-half hours after the laying of the
previous laid egg, the transgene is microinjected into the
cytoplasm of the germinal disc and the embryo is cultured in a host
shell until maturity. When the animals to be made transgenic are
bovine or porcine, microinjection can be hampered by the opacity of
the ova thereby making the nuclei difficult to identify by
traditional differential interference-contrast microscopy. To
overcome this problem, the ova can first be centrifuged to
segregate the pronuclei for better visualization.
[0194] The non-human transgenic animals of the invention include,
for example, bovine, porcine, ovine and avian animals (e.g., cow,
pig, sheep, chicken, turkey). Such transgenic non-human animals are
produced by introducing a transgene into the germline of the
non-human animal. Embryonal target cells at various developmental
stages can be used to introduce transgenes. Different methods are
used depending on the stage of development of the embryonal target
cell. The zygote is the best target for microinjection. The use of
zygotes as a target for gene transfer has a major advantage in that
in most cases the injected DNA will be incorporated into the host
gene before the first cleavage (Brinster et al., Proc. Natl. Acad.
Sci. USA 82:4438-4442, 1985). As a consequence, all cells of the
transgenic non-human animal will carry the incorporated transgene.
This will in general also be reflected in the efficient
transmission of the transgene to offspring of the founder since 50%
of the germ cells will harbor the transgene.
[0195] The term "transgenic" is used to describe an animal that
includes exogenous genetic material within all of its cells. A
transgenic animal can be produced by cross-breeding two chimeric
animals that include exogenous genetic material within cells used
in reproduction. Twenty-five percent of the resulting offspring
will be transgenic, i.e., animals that include the exogenous
genetic material within all of their cells in both alleles. 50% of
the resulting animals will include the exogenous genetic material
within one allele and 25% will include no exogenous genetic
material.
[0196] In the microinjection method useful in the practice of the
invention, the transgene is digested and purified free from any
vector DNA, e.g., by gel electrophoresis. It is preferred that the
transgene include an operatively associated promoter that interacts
with cellular proteins involved in transcription, ultimately
resulting in constitutive expression. Promoters useful in this
regard include those from cytomegalovirus (CMV), Moloney leukemia
virus (MLV), and herpes virus, as well as those from the genes
encoding metallothionein, skeletal actin, P-enolpyruvate
carboxylase (PEPCK), phosphoglycerate (PGK), DHFR, and thymidine
kinase. Promoters for viral long terminal repeats (LTRs) such as
Rous Sarcoma Virus can also be employed. When the animals to be
made transgenic are avian, preferred promoters include those for
the chicken .theta.-globin gene, chicken lysozyme gene, and avian
leukosis virus. Constructs useful in plasmid transfection of
embryonic stem cells will employ additional regulatory elements
well known in the art such as enhancer elements to stimulate
transcription, splice acceptors, termination and polyadenylation
signals, and ribosome binding sites to permit translation.
[0197] Retroviral infection can also be used to introduce transgene
into a non-human animal, as described above. The developing
non-human embryo can be cultured in vitro to the blastocyst stage.
During this time, the blastomeres can be targets for retro viral
infection (Jaenich, Proc. Natl. Acad. Sci. USA 73:1260-1264, 1976).
Efficient infection of the blastomeres is obtained by enzymatic
treatment to remove the zona pellucida (Hogan et al., In
"Manipulating the Mouse Embryo" (Cold Spring Harbor Laboratory
Press, Cold Spring Harbor N.Y. 1986)). The viral vector system used
to introduce the transgene is typically a replication-defective
retro virus carrying the transgene (Jahner et al, Proc. Natl. Acad.
Sci. USA 82:6927-6931, 1985; Van der Putten et al., Proc. Natl.
Acad. Sci USA 82:6148-6152, 1985). Transfection is easily and
efficiently obtained by culturing the blastomeres on a monolayer of
virus-producing cells (Van der Putten, supra; Stewart, et al., EMBO
J. 6:383-388, 1987). Alternatively, infection can be performed at a
later stage. Virus or virus-producing cells can be injected into
the blastocoele (Jahner et al., Nature 298:623-628, 1982). Most of
the founders will be mosaic for the transgene since incorporation
occurs only in a subset of the cells that formed the transgenic
nonhuman animal. Further, the founder can contain various
retroviral insertions of the transgene at different positions in
the genome that generally will segregate in the offspring. In
addition, it is also possible to introduce transgenes into the germ
line, albeit with low efficiency, by intrauterine retroviral
infection of the midgestation embryo (Jahner et al., supra,
1982).
[0198] A third type of target cell for transgene introduction is
the embryonal stem cell (ES). ES cells are obtained from
pre-implantation embryos cultured in vitro and fused with embryos
(Evans et al. Nature 292:154-156, 1981; Bradley et al., Nature
309:255-258, 1984; Gossler et al., Proc. Natl. Acad. Sci. USA
83:9065-9069, 1986; and Robertson et al., Nature 322:445-448,
1986). Transgenes can be efficiently introduced into the ES cells
by DNA transfection or by retro virus-mediated transduction. Such
transformed ES cells can thereafter be combined with blastocysts
from a nonhuman animal. The ES cells thereafter colonize the embryo
and contribute to the germ line of the resulting chimeric animal
(for review see Jaenisch, Science 240:1468-1474, 1988).
[0199] The transgene can be any piece of DNA that is inserted by
artifice into a cell, and becomes part of the genome of the
organism (i.e., either stably integrated or as a stable
extrachromosomal element) that develops from that cell. Such a
transgene can include a gene that is partly or entirely
heterologous (i.e., foreign) to the transgenic organism, or can
represent a gene homologous to an endogenous gene of the organism.
Included within this definition is a transgene created by the
providing of an RNA sequence that is transcribed into DNA, then
incorporated into the genome. The transgenes of the invention
include DNA sequences that encode a mutant PKD1 polypeptide, for
example, a polypeptide having an amino acid sequence substantially
identical to SEQ ID NO:2 and having a mutation of a A88V, a W967R,
a L2696R, an R2985G, an R3039C, a V3285I, a H3311R, or any
combination thereof; or encoding a truncated PKD1 polypeptide
ending at amino acid 3000 (also referred to herein as "W3001X",
where "X" indicates STOP codon; see, also, Table 4) and include
sense, antisense, and dominant negative encoding polynucleotides,
which can be expressed in a transgenic non-human animal. The term
"transgenic" as used herein also includes any organism whose genome
has been altered by in vitro manipulation of the early embryo or
fertilized egg or by any transgenic technology to induce a specific
gene knockout. The term "gene knockout" as used herein, refers to
the targeted disruption of a gene in vivo with complete or partial
loss of function that has been achieved by any transgenic
technology familiar to those in the art. In one embodiment,
transgenic animals having a gene knockout are those in which the
target gene has been rendered nonfunctional by an insertion
targeted to the gene to be rendered non-functional by homologous
recombination.
[0200] The invention also includes animals having heterozygous
mutations in or partial inhibition of function or expression of a
PKD1 polypeptide. One of skill in the art would readily be able to
determine if a particular mutation or if an antisense molecule was
able to partially inhibit PKD1 expression. For example, in vitro
testing can be desirable initially by comparison with wild-type
(e.g., comparison of northern blots to examine a decrease in
expression). After an embryo has been microinjected, colonized with
transfected embryonic stem cells or infected with a retrovirus
containing the transgene (except for practice of the subject
invention in avian species, which is addressed elsewhere herein),
the embryo is implanted into the oviduct of a pseudopregnant
female. The progeny are tested for incorporation of the transgene
by Southern blot analysis of blood samples using transgene specific
probes. PCR is particularly useful in this regard. Positive progeny
(P.sub.0) are crossbred to produce offspring (P.sub.1) that are
analyzed for transgene expression by northern blot analysis of
tissue samples.
[0201] In order to distinguish expression of like species
transgenes from expression of an endogenous PKD1-related gene, a
marker gene fragment can be included in the construct in the 3'
untranslated region of the transgene and the northern blot probe
designed to probe for the marker gene fragment. The serum levels of
a PKD1 polypeptide can also be measured in the transgenic animal to
determine the level of PKD1 expression. A method of creating a
transgenic organism also can include methods of inserting a
transgene into, for example, an embryo of an already created
transgenic organism, the organism being transgenic for a different
unrelated gene or polypeptide.
[0202] Transgenic organisms of the invention are highly useful in
the production of organisms for study of, for example, polycystic
kidney disease or PKD1-related diseases or disorders and in
identifying agents or drugs that inhibit or modulate polycystic
kidney disease, PKD1 associated disorders and inheritance.
Expression of a mutant human PKD1 polynucleotide can be assayed,
for example, by standard northern blot analysis, and the production
of the mutant human PKD1 polypeptide can be assayed, for example,
by detecting its presence using an antibody directed against the
mutant human PKD1 polypeptide. Those animals found to express the
mutant human PKD1 polypeptide can then be observed for the
development of ADPKD-like symptoms.
[0203] As discussed above, animal models of ADPKD can be produced
by engineering animals containing mutations in a copy of an
endogenous PKD1 gene that correspond to mutations within the human
PKD1 polynucleotide. Utilizing such a strategy, a PKD1 homologue
can be identified and cloned from the animal of interest, using
techniques such as those described herein. One or more mutations
can be engineered into such a PKD1 homologue that correspond to
mutations within the human PKD1 polynucleotide, as discussed above
(e.g., resulting in a mutation of the amino acid sequence as set
forth in SEQ ID NO:2 and having a mutation of a A88V, a W967R, a
L2696R, an R2985G, a W3001X, an R3039C, a V3285I, a H3311R, or any
combination thereof; see, also, Table 4). As disclosed herein, a
mutant polypeptide produced by such an engineered corresponding
PKD1 homologue can exhibit an aberrant PKD1 activity that is
substantially similar to that exhibited by a mutant human PKD1
protein. The engineered PKD1 homologue can then be introduced into
the genome of the animal of interest, using techniques such as
those described, above. Accordingly, any of the ADPKD animal models
described herein can be used to test compounds for an ability to
ameliorate ADPKD symptoms, including those associated with the
expression of a mutant PKD1 polypeptide substantially identical to
SEQ ID NO:2 and having the mutation A88V, W967R, L2696R, R2985G,
W3001X, R3039C, V3285I, H3311R, or a combination thereof (see
Example 2 and Table 4).
[0204] As discussed above, mutations in the PKD1 polynucleotide
that cause ADPKD can produce a form of the PKD1 protein that
exhibits an aberrant activity that leads to the formation of ADPKD
symptoms. A variety of techniques can be utilized to inhibit the
expression, synthesis, or activity of such mutant PKD1
polynucleotides and polypeptides. For example, compounds such as
those identified through assays described, above, which exhibit
inhibitory activity, can be used in accordance with the invention
to ameliorate ADPKD symptoms. Such molecules can include, but are
not limited, to small and large organic molecules, peptides, and
antibodies. Further, antisense and ribozyme molecules that inhibit
expression of a PKD1 polynucleotide, (e.g., a mutant PKD1
polynucleotide), can also be used to inhibit the aberrant PKD1
activity. Such techniques are described, below. In yet another
embodiment, triple helix molecules can be utilized in inhibiting
aberrant PKD1 activity.
[0205] Among the compounds that can exhibit anti-ADPKD activity are
antisense, ribozyme, and triple helix molecules. Such molecules can
be designed to reduce or inhibit mutant PKD1 activity by modulating
the expression or synthesis of PKD1 polypeptides. Techniques for
the production and use of such molecules are well known to those of
skill in the art.
[0206] Double stranded interfering RNA molecules are especially
useful to inhibit expression of a target gene. For example, double
stranded RNA molecules can be injected into a target cell or
organism to inhibit expression of a gene and the resultant
polypeptide's activity. It has been found that such double stranded
RNA molecules are more effective at inhibiting expression than
either RNA strand alone (Fire et al., Nature, 19:391(6669):806-11,
1998).
[0207] When a disorder is associated with abnormal expression of a
PKD1 polypeptide (e.g., overexpression, or expression of a mutated
form of the protein), a therapeutic approach that directly
interferes with the translation of a PKD1 polypeptide (e.g., a wild
type, variant or mutant PKD1 polypeptide) is possible.
Alternatively, similar methodology can be used to study gene
activity. For example, antisense nucleic acid, double stranded
interfering RNA or ribozymes could be used to bind to a PKD1 mRNA
sequence or to cleave it. Antisense RNA or DNA molecules bind
specifically with a targeted gene's RNA message, interrupting the
expression of that gene's protein product. The antisense binds to
the messenger RNA forming a double stranded molecule that cannot be
translated by the cell. Antisense oligonucleotides of about 15 to
25 nucleotides are preferred since they are easily synthesized and
have an inhibitory effect just like antisense RNA molecules. In
addition, chemically reactive groups, such as iron-linked
ethylenediaminetetraacetic acid (EDTA-Fe) can be attached to an
antisense oligonucleotide, causing cleavage of the RNA at the site
of hybridization. Antisense nucleic acids are DNA or RNA molecules
that are complementary to at least a portion of a specific mRNA
molecule (Weintraub, Scientific American, 262:40, 1990). In the
cell, the antisense nucleic acids hybridize to the corresponding
mRNA, forming a double-stranded molecule. The antisense nucleic
acids interfere with the translation of the mRNA, since the cell
will not translate a mRNA that is double-stranded. Antisense
oligomers of at least about 15 nucleotides also are preferred
because they are less likely to cause problems when introduced into
the target PKD1 polypeptide producing cell. The use of antisense
methods to inhibit the in vitro translation of genes is well known
in the art (Marcus-Sakura, Anal. Biochem., 172:289, 1988).
[0208] Use of an oligonucleotide to stall transcription is known as
the triplex strategy since the oligomer winds around double-helical
DNA, forming a three-strand helix. Therefore, these triplex
compounds can be designed to recognize a unique site on a chosen
gene (Maher et al., Antisense Res. and Devel., 1:227, 1991; Helene,
Anticancer Drug Design, 6:569, 1991).
[0209] Ribozymes are RNA molecules possessing the ability to
specifically cleave other single-stranded RNA in a manner analogous
to DNA restriction endonucleases. Through the modification of
nucleotide sequences that encode these RNAs, it is possible to
engineer molecules that recognize specific nucleotide sequences in
an RNA molecule and cleave it (Cech, J. Amer. Med. Assn., 260:3030,
1988). A major advantage of this approach is that, because they are
sequence-specific, only mRNAs with particular sequences are
inactivated.
[0210] There are two basic types of ribozymes namely,
tetrahymena-type (Hasselhoff, Nature, 334:585, 1988) and
"hammerhead"-type. Tetrahymena-type ribozymes recognize sequences
that are four bases in length, while "hammerhead"-type ribozymes
recognize base sequences 11-18 bases in length. The longer the
recognition sequence, the greater the likelihood that the sequence
will occur exclusively in the target mRNA species. Consequently,
hammerhead-type ribozymes are preferable to tetrahymena-type
ribozymes for inactivating a specific mRNA species and 18-base
recognition sequences are preferable to shorter recognition
sequences. These and other uses of antisense and ribozymes methods
to inhibit the in vivo translation of genes are known in the art
(e.g., De Mesmaeker et al, Curr. Opin. Struct. Biol., 5:343, 1995;
Gewirtz et al., Proc. Natl. Acad. Sci. USA, 93:3161, 1996b; Stein,
Chem. and Biol. 3:319, 1996).
[0211] Specific ribozyme cleavage sites within any potential RNA
target are initially identified by scanning the target molecule for
ribozyme cleavage sites, which include the following sequence: GUA,
GUU and GUC. Once identified, short RNA sequences of about 15 to 30
ribonucleotides corresponding to the region of the target gene
containing the cleavage site can be evaluated for predicted
structural features, such as secondary structure, that can render
the oligonucleotide sequence unsuitable. The suitability of
candidate targets can also be evaluated by testing their
accessibility to hybridization with complementary oligonucleotides,
using ribonuclease protection assays.
[0212] It is possible that the antisense, ribozyme, or triple helix
molecules described herein can reduce or inhibit the translation of
mRNA produced by mutant PKD1 alleles of the invention. In order to
ensure that substantial normal levels of PKD1 activity are
maintained in the cell, nucleic acid molecules that encode and
express PKD1 polypeptides exhibiting normal PKD1 activity can be
introduced into cells that do not contain sequences susceptible to
whatever antisense, ribozyme, or triple helix treatments. Such
sequences can be introduced via gene therapy methods such as those
described, below. Alternatively, it can be preferable to
coadminister normal PKD1 protein into the cell or tissue in order
to maintain the requisite level of cellular or tissue PKD1
activity.
[0213] Antisense RNA and DNA molecules, ribozyme molecules and
triple helix molecules of the invention can be prepared by any
method known in the art for the synthesis of DNA and RNA molecules.
These include techniques for chemically synthesizing
oligodeoxyribonucleotides and oligoribonucleotides well known in
the art such as for example solid phase phosphoramidite chemical
synthesis. Alternatively, RNA molecules can be generated by in
vitro and in vivo transcription of DNA sequences encoding the
antisense RNA molecule. Such DNA sequences can be incorporated into
a wide variety of vectors that incorporate suitable RNA polymerase
promoters such as the T7 or SP6 polymerase promoters.
Alternatively, antisense cDNA constructs that synthesize antisense
RNA constitutively or inducibly, depending on the promoter used,
can be introduced stably into cell lines.
[0214] Various well known modifications to the DNA molecules can be
introduced as a means of increasing intracellular stability and
half-life. Possible modifications include, but are not limited to,
the addition of flanking sequences of ribonucleotide or
deoxyribonucleotides to the 5' or 3' end or both of the molecule or
the use of phosphorothioate or 2'-O-methyl rather than
phosphodiesterase linkages within the oligodeoxyribonucleotide
backbone.
[0215] As discussed above, mutations in the PKD1 polynucleotide
that cause ADPKD can lower the level of expression of the PKD1
polynucleotide or; alternatively, can cause inactive or
substantially inactive PKD1 proteins to be produced. In either
instance, the result is an overall lower level of normal PKD1
activity in the tissues or cells in which PKD1 is normally
expressed. This lower level of PKD1 activity, then, leads to ADPKD
symptoms. Thus, such PKD1 mutations represent dominant
loss-of-function mutations. For example, a polynucleotide having a
sequence as set forth in SEQ ID NO:1 and having a mutation of a
G9213A results in early termination of PKD1.
[0216] For example, normal PKD1 protein, at a level sufficient to
ameliorate ADPKD symptoms can be administered to a patient
exhibiting such symptoms or having a mutant PKD1 polynucleotide.
Additionally, DNA sequences encoding normal PKD1 protein can be
directly administered to a patient exhibiting ADPKD symptoms or
administered to prevent or reduce ADPKD symptoms where they have
been diagnosed as having a PKD1 mutation identified herein but have
not yet demonstrated symptoms. Such administration can be at a
concentration sufficient to produce a level of PKD1 protein such
that ADPKD symptoms are ameliorated.
[0217] Further, subjects with these types of mutations can be
treated by gene replacement therapy. A copy of the normal PKD1
polynucleotide can be inserted into cells, renal cells, for
example, using viral or non-viral vectors that include, but are not
limited to vectors derived from, for example, retroviruses,
vaccinia virus, adeno-associated virus, herpes viruses, bovine
papilloma virus or non-viral vectors, such as plasmids. In
addition, techniques frequently employed by those skilled in the
art for introducing DNA into mammalian cells can be utilized. For
example, methods including but not limited to electroporation,
DEAE-dextran mediated DNA transfer, DNA guns, liposomes, direct
injection, and the like can be utilized to transfer recombinant
vectors into host cells. Alternatively, the DNA can be transferred
into cells through conjugation to proteins that are normally
targeted to the inside of a cell. For example, the DNA can be
conjugated to viral proteins that normally target viral particles
into the targeted host cell.
[0218] Administering the whole gene or polypeptide is not necessary
to avoid the appearance of ADPKD symptoms. The use of a "minigene"
therapy approach also can serve to ameliorate such ADPKD symptoms
(see Ragot et al., Nature 3:647, 1993; Dunckley et al., Hum. Mol.
Genet. 2:717-723, 1993). A minigene system uses a portion of the
PKD1 coding region that encodes a partial, yet active or
substantially active PKD1 polypeptide. As used herein,
"substantially active" means that the polypeptide serves to
ameliorate ADPKD symptoms. Thus, the minigene system utilizes only
that portion of the normal PKD1 polynucleotide that encodes a
portion of the PKD1 polypeptide capable of ameliorating ADPKD
symptoms, and can, therefore represent an effective and even more
efficient ADPKD therapy than full-length gene therapy approaches.
Such a minigene can be inserted into cells and utilized via the
procedures described, above, for full-length gene replacement. The
cells into which the PKD1 minigene are to be introduced are,
preferably, those cells, such as renal cells, which are affected by
ADPKD. Alternatively, any suitable cell can be transfected with a
PKD1 minigene so long as the minigene is expressed in a sustained,
stable fashion and produces a polypeptide that ameliorates ADPKD
symptoms.
[0219] A therapeutic minigene for the amelioration of ADPKD
symptoms can comprise a nucleotide sequence that encodes at least
one PKD1 polypeptide peptide domain, particularly a domain having
an amino acid sequence substantially identical to a peptide portion
SEQ ID NO:2 and having a mutation as shown in Table 4, for example,
an A88V, W967R, L2696R, R2985G, W3001X, R3039C, V3285I, or H3311R
mutation. Minigenes that encode such PKD1 polypeptides can be
synthesized and/or engineered using the PKD1 polynucleotide
sequence (SEQ ID NO:1).
[0220] The materials for use in the assay of the invention are
ideally suited for the preparation of a kit. Such a kit can
comprise a carrier means containing one or more container means
such as vials, tubes, and the like, each of the container means
comprising one of the separate elements to be used in the method.
One of the container means can comprise a probe that is or can be
detectably labeled. Such probe can be an oligonucleotide comprising
at least 10 contiguous nucleotides and having a sequence of a
fragment of SEQ ID NO:1 including: nucleotide 474, wherein
nucleotide 474 is a T; nucleotide 487, wherein nucleotide 487 is an
A; nucleotide 3110, wherein nucleotide 3110 is a C; nucleotide
8298, wherein nucleotide 8298 is a G; nucleotide 9164, wherein
nucleotide 9164 is a G; nucleotide 9213, wherein nucleotide 9213 is
an A; nucleotide 9326, wherein nucleotide 9326 is a T; nucleotide
9367, wherein nucleotide 9367 is a T; nucleotide 10064, wherein
nucleotide 10064 is an A; nucleotide 10143, wherein nucleotide
10143 is a G; nucleotide 10234, wherein nucleotide 10234 is a C; or
nucleotide 10255, wherein nucleotide 10255 is a T (see, also,
Example 2).
[0221] A kit containing one or more oligonucleotide probes of the
invention can be useful, for example, for qualitatively identifying
the presence of mutant PKD1 polynucleotide sequences in a sample,
as well as for quantifying the degree of binding of the probe for
determining the occurrence of specific strongly binding
(hybridizing) sequences, thus indicating the likelihood for a
subject having or predisposed to a disorder associated with PKD1.
Where the kit utilizes nucleic acid hybridization to detect the
target nucleic acid, the kit can also have containers containing
reagents for amplification of the target nucleic acid sequence.
When it is desirable to amplify the mutant target sequence, this
can be accomplished using oligonucleotide primers, which are based
upon identification of the flanking regions contiguous with the
target nucleotide sequence. For example, primers such as those
listed below in Tables 1 and 2 can be included in the kits of the
invention. The kit can also contain a container comprising a
reporter means such as an enzymatic, fluorescent, or radionuclide
label, which can be bound to or incorporated into the
oligonucleotide and can facilitate identification of the
oligonucleotide.
[0222] The following examples are intended to illustrate but not
limit the invention.
EXAMPLES
[0223] The present invention is based upon the use of widely spaced
PKD1-specific anchor primers in long range PCR to generate 5 kb to
10 kb PKD1 polynucleotide segments. After appropriate dilution, the
PCR products can be used as a template for mutation screening using
any one of a variety of methods. Accordingly, a number of mutants
have been identified in families with PKD1-associated
disorders.
[0224] Using a number of PKD1-specific primers, eight templates
ranging in size from about 0.3 to 5.8 kb were generated that span
from the 5' untranslated region to intron 34 and cover all exons in
the replicated region including exon 1 and exon 22 (Example 1).
These reagents were used to evaluate 47 Asian PKD1 families
(Example 2). Variant nucleotide sequences were found throughout the
PKD1 polynucleotide sequence.
[0225] Forty-one Thai and 6 Korean ADPKD families were studied.
Samples from 50 healthy Thai blood donors collected in blood banks
served as normal controls. Genomic DNA was extracted from either
fresh or frozen whole blood that had been stored for up to five
years using commercially available kits (Puregene, Gentra) or
standard phenol-chloroform methods. For the N23HA and 145.19 cell
lines (Cell 77:881-894, 1994; Germino et al., Am J. Hum. Genet.
46:925-933, 1990; Ceccherini et al., Proc. Natl. Acad. Sci. USA
89:104-108, 1992, each of which is incorporated herein by
reference; see, also, Watnick et al., supra, 1997), genomic DNA was
isolated using the Puregene DNA isolation kit.
Example 1
Long Range Specific Templates
[0226] A two-part strategy was used to generate and validate
PKD1-specific primers that could be used to amplify the replicated
portion of PKD1. The sequence of PKD1 (SEQ ID NO:1) was aligned
with that of two homologues present in GenBank (Accession Number
AC002039) and identified potential sequence differences. Candidate
primers were designed such that the mismatches were positioned at
or adjacent to the 3' end of the oligonucleotide so as to maximize
their specificity for PKD1.
[0227] The primers were tested for specificity using rodent-human
somatic cell hybrids that either contained only human 16p13.3 and
therefore, human PKD1 (145.19, a radiation hybrid), or that lacked
16p13.3 and contained only the human PKD1-homologues (N23HA). FIG.
2 presents a representative example of this approach using the
primer pair, BPF6 and the PKD1-specific primer BPR6. This primer
pair amplified a product of the correct length (4.5 kb) under the
stated conditions only when total human genomic DNA or 145.19 DNA
is used as template. Similar results were obtained when BPR6 was
used in combination with the non-specific primer 28F to generate a
much shorter product.
[0228] As a final control, the absence of amplified product was
verified using N23HA as template to confirm that the results
obtained using total human genomic DNA and 145.19 DNA were due to
the specificity of the primer and not the result of other causes
(i.e., difference in quality of DNA or ratio of human/rodent
template). A primer specific for the homologues (BPR6HG) was
designed that was positioned the same distance from BPF6 as BPR6
and used to amplify a specific band of the same size as the
corresponding PKD1-long range product. As predicted, a product of
the correct size was amplified from both N23HA and total genomic
DNA, but not from 145.19.
[0229] A total of eight primer pairs can be used to generate a
series of templates that range in size from about 0.3 kb to 5.8 kb
and include all exons and their flanking intron sequences in the
replicated portion of PKD1 (exons 1 to 34). Table 1 summarizes the
details for each product and includes the sequence of each primer,
its respective position within the gene, its expected size, and the
optimal annealing temperature and extension time for its
amplification. FIG. 1 illustrates the relative position of each
product with respect to the overall gene structure. It should be
noted that exon 1 and its flanking sequences were particularly
problematic to evaluate. Primer design was greatly limited by the
high degree of homology and extreme GC bias in the region. A
combination of widely space primers (to generate a fragment
considerably larger than the segment of interest) and the GC melt
system were used to circumvent these obstacles.
[0230] Specific details concerning the primer sequences, annealing
temperatures and extension times used for each long-range (LR)
template are provided in Table 1 (all sequences in Tables 1 and 2
are shown in 5' to 3' orientation from left to right). Three
hundred to 400 ng of genomic DNA was used as template for each LR
product, except for exon 1 (see below). The long range PCR
amplification was performed as follows in a Perkin Elmer 9600
thermal cycler: denaturation at 95.degree. C. for 3 min followed by
35 cycles of a two-step protocol that included denaturation at
95.degree. C. for 20 sec followed by annealing and extension at a
temperature and for a time specific for each primer pair (Table 1).
A final extension at 72.degree. C. for 10 min was included in each
program. The total PCR volume was 50 Tl using 4 U of rTth DNA
polymerase XL (Cetus, Perkin Elmer) and a final MgOAC.sub.2
concentration of 0.9 mM. A hot start protocol as recommended by the
manufacturer was used for the first cycle of amplification. For the
exon 1 LR product (T1), the LR was generated using 500 ng of
genomic DNA. The long range PCR amplification was modified as
follows: denaturation 95.degree. C. for 1 min followed by 35
two-step cycles of denaturation at 95.degree. C. for 30 sec
followed by annealing and extension at 69.degree. C. for 7 min. The
total PCR volume was 50 Tl using 1 Tl of Advantage-GC genomic
polymerase (Clontech), GC melt of 1.5 M and final MgOAC.sub.2
concentration of 1.1 mM.
TABLE-US-00001 TABLE 1 Oligonucleotide Primers for Long-Range
Specific Templates from Exon 1-34 of PKD1 Gene SEQ Position Size Tm
ET ID Template Primers Sequence 5'.quadrature.3' (5') (kb)
(.degree. C.) (Min) NO: T1 BPF14* CCATCCACCTGCTGTGTGAC 2043 2.2 69
7 3 CTGGTAAAT BPR9 CCACCTCATCGCCCCTTCCT 4290 4 AAGCAT T2-7 BPF9*
ATTTTTTGAGATGGAGCTTC 17907 4.6 68 7 5 ACTCTTGCAGG BPR4
CGCTCGGCAGGCCCCTAACC 22489 6 T8-12 BPF12 CCGCCCCCAGGAGCCTAGACG
22218 4.2 68 7 7 BPR5* CATCCTGTTCATCCGCTCCA 26363 8 CGGTTAC T13-15
F13 TGGAGGGAGGGACGCCAATC 26246 4.4 68 7 9 R27* GTCAACGTGGGCCTCCAAGT
30612 10 T15-21 F26* AGCGCAACTACTTGGAGGCCC 30603 3.4 70 4.5 11 R2
GCAGGGTGAGCAGGTGGGG 33953 12 CCATCCTAC T22 BPF15
GAGGCTGTGGGGGTCCAGTC 36815 0.3 72 1 13 AAGTGG BPR12*
AGGGAGGCAGAGGAAAGGG 37136 14 CCGAAC T23-28 BPF6
CCCCGTCCTCCCCGTCCTTTT 37325 4.2 69 7 15 GTC BPR6*
AAGCGCAAAAGGGCTGCGT 41524 16 CG T29-34 BPF13* GGCCCTCCCTGCCTTCTAGG
41504 5.8 68 8 17 CG KG8R25* GTTGCAGCCAAGCCCATGTTA 47316 18
Tm--annealing temperature; ET--extension time; *PKD1-specific
primer. Bold type in BPR12 primer sequence identifies intentional
replacement of C by A to enhance discrimination of PKD1 from
homologs.
[0231] The long-range templates were serially diluted (1:10.sup.4
or 1:10.sup.5) to remove genomic contamination, then used as
templates for nested PCR of 200-400 bp exonic fragments. A total of
17 new primer pairs were developed for exons 1-12 and exon 22. The
sequences and PCR conditions for each new pair are summarized in
Table 2. Primer sequences and PCR conditions for exons 13-21 and
23-34 are described in Watnick et al., Am. J. Hum. Genet.
65:1561-1571, 1999; and Watnick et al., Hum. Mol. Genet.
6:1473-1481, 1997, which are incorporated herein by reference.
Intron based primers were positioned approximately 30-50 bp away
from consensus splice sites. Exons larger than approximately 400 bp
were split into overlapping fragments of less than or equal to 350
bp. Two Tl of diluted long range (LR) product was used as template
for amplification of each exon. Single strand conformation analysis
was performed using standard protocols. SSCA analysis was performed
by use of 8% polyacrylamide gels with 5% glycerol added. The
radiolabeled PCR products were diluted with loading buffer, were
denatured by heating at 95.degree. C. for 5 min, then were placed
on ice prior to being loaded and run on the gel at room
temperature. Gels were run at 400 V overnight, dried, and placed on
X-Omat XAR film (Kodak) at room temperature. Aberrantly migrating
bands detected by SSCA were cut from the gel and eluted into 100 Tl
of sterile water overnight. The eluted products were re-amplified
using the same set of primers, purified using Centricon-100 columns
(Amicon) and then sequences.
TABLE-US-00002 TABLE 2 Nested Primers Used for Mutation Detection
Fragment SEQ size Tm ID Exons Primer Primer Sequence 5'.fwdarw.3'
(bp) (.degree. C.) NO: T1 1F1 GGTCGCGCTGTGGCGAAGG 328 67 19 T1 1R1
CGGCGGGCGGCATCGT 20 T1 1F2 ACGGCGGGGCCATGCG 348 67 21 T1 1R2
GCGTCCTGGCCCGCGTCC 22 T2-7 2F TTGGGGATGCTGGCAATGTG 272 62 23 T2-7
2R GGGATTCGGCAAAGCTGATG 24 T2-7 3F CCATCAGCTTTGCCGAATCC 171 62 25
T2-7 3R AGGGCAGAAGGGATATTGGG 26 T2-7 4F AGACCCTTCCCACCAGACCT 299 62
27 T2-7 4R TGAGCCCTGCCCAGTGTCT 28 T2-7 5F1 GAGCCAGGAGGAGCAGAACCC
259 65 29 T2-7 5R1 AGAGGGACAGGCAGGCAAAGG 30 T2-7 5F2
CCCAGCCCTCCAGTGCCT 284 65 31 T2-7 5R2 CCCAGGCAGCACATAGCGAT 32 T2-7
5F3 CCGAGGTGGATGCCGCTG 294 65 33 T2-7 5R3 GAAGGGGAGTGGGCAGCAGAC 34
T2-7 6F CACTGACCGTTGACACCCTCG 281 65 35 T2-7 6R
TGCCCCAGTGCTTCAGAGATC 36 T2-7 7F GGAGTGCCCTGAGCCCCCT 311 65 37 T2-7
7R CCCCTAACCACAGCCAGCG 38 T8-12 8F TCTGTTCGTCCTGGTGTCCTG 215 65 39
T8-12 8R GCAGGAGGGCAGGTTGTAGAA 40 T8-12 9F GGTAGGGGGAGTCTGGGCTT 253
65 41 T8-12 9R GAGGCCACCCCGAGTCC 42 T8-12 10F GTTGGGCATCTCTGACGGTG
364 65 43 T8-12 10R GGAAGGTGGCCTGAGGAGAT 44 T8-12 11F2
GGGGTCCACGGGCCATG 311 67 45 T8-12 11R2 AAGCCCAGCAGCACGGTGAG 46
T8-12 11midF GCTTGCAGCCACGGAAC 386 65 47 T8-12 11midR
GCAGTGCTACCACTGAGAAC 48 T8-12 11F1 TGCCCCTGGGAGACCAACGATAC 303 67
49 T8-12 11R1 GGCTGCTGCCCTCACTGGGAAG 50 12 12F GAGGCGACAGGCTAAGGG
286 64 51 12R-2 CATGAAGCAGAGCAGAAGG 61 13 13F: TGGAGGGAGGGACGCCAATC
308 67 62 13R: GAGGCTGGGGCTGGGACAA 63 14 14F: CCCGGTTCACTCACTGCG
220 64 64 14R: CCGTGCTCAGAGCCTGAAAG 65 15 15F16: CGGGTGGGGAGCAGGTGG
280 67 66 15R16: GCTCTGGGTCAGGACAGGGGA 67 15 15F15:
CGCCTGGGGGTGTTCTTT 270 64 68 15R15: ACGTGATGTTGTCGCCCG 69 15 15F14:
GCCCCCGTGGTGGTCAGC 250 67 70 15R14: CAGGCTGCGTGGGGATGC 71 15 15F13:
CTGGAGGTGCTGCGCGTT 256 67 72 15R13: CTGGCTCCACGCAGATGC 73 15 15F12:
CGTGAACAGGGCGCATTA 270 65 74 15R12: GCAGCAGAGATGTTGTTGGAC 75 15
15F11: CCAGGCTCCTATCTTGTGACA 259 60 76 15R11: TGAAGTCACCTGTGCTGTTGT
77 15 15F10: CTACCTGTGGGATCTGGGG 217 67 78 15R10:
TGCTGAAGCTCACGCTCC 79 15 15F9: GGGCTCGTCGTCAATGCAAG 267 67 80 15R9:
CACCACCTGCAGCCCCTCTA 81 15 15F8: 5CCGCCCAGGACAGCATCTTC 261 64 82
15R8: CGCTGCCCAGCATGTTGG 83 15 15F7: CGGCAAAGGCTTCTCGCTC 288 64 84
15R7: CCGGGTGTGGGGAAGCTATG 85 15 15F6: CGAGCCATTTACCACCCATAG 231 65
86 15R6: GCCCAGCACCAGCTCACAT 87 15 15F5: CCACGGGCACCAATGTGAG 251 64
88 15R5: GGCAGCCAGCAGGATCTGAA 89 15 15F4: CAGCAGCAAGGTGGTGGC 333 67
90 15R4: GCGTAGGCGACCCGAGAG 91 15 15F3: ACGGGCACTGAGAGGAACTTC 206
64 92 15R3: ACCAGCGTGCGGTTCTCACT 93 15 15F2: GCCGCGACGTCACCTACAC
265 67 94 15R2: TCGGCCCTGGGCTCATCT 95 15 15F1: GTCGCCAGGGCAGGACACAG
228 68 96 R27': AGGTCAACGTGGGCCTCCAA 113 15 15F1-1:
ACTTGGAGGCCCACGTTGACC 276 69 97 15R1-1: TGATGGGCACCAGGCGCTC 98 15
15F1-2: CATCCAGGCCAATGTGACGGT 266 64 99 15R1-2:
CCTGGTGGCAAGCTGGGTGTT 100 16 16F: TAAAACTGGATGGGGCTCTC 294 56 101
16R: GGCCTCCACCAGCACTAA 102 17 17F: GGGTCCCCCAGTCCTTCCAG 244 67 103
17R: TCCCCAGCCCGCCCACA 104 18 18F: GCCCCCTCACCACCCCTTCT 342 67 105
18R: TCCCGCTGCTCCCCCCAC 106 19 19F: GATGCCGTGGGGACCGTC 285 67 107
19R: GTGAGCAGGTGGCAGTCTCG 108 20 20F: CCACCCCCTCTGCTCGTAGGT 232 64
109 20R: GGTCCCAAGCACGCATGCA 110 21 21F: TGCCGGCCTCCTGCGCTGCTGA 232
67 111 TWR2-1: GTAGGATGGCCCCACCTGCTCAC 112 CCTGC
[0232] Variants that were predicted to alter a restriction site
were confirmed by restriction enzyme digestion analysis of
re-amplified products. In cases where the change did not alter a
restriction site, primers were designed with mismatches that create
a new restriction site when combined with the point mutation in
question. The following primer combinations were utilized:
TABLE-US-00003 ASP1 + 26R (ASP1; 5'-CTGGTGACCTACATGGTCATGGCC
GAGATC-3'; SEQ ID NO: 55); ASP2 + 30R (ASP2;
5'-GGTTGTCTATCCCGTCTACCTGGC CCTCCT-3'; SEQ ID NO: 56); ASP3 + 30F
(ASP3; 5'-GTCCCCAGCCCCAGCCCACCTGGC C-3'; SEQ ID NO: 57).
[0233] When possible, segregation of the variant with the disease
phenotype was tested. In cases where a missense change was unable
to be determined on the normal haplotype (and thus be a normal
variant) the mutation was tested for in a panel of 50 normal
controls.
Example 2
Mutation Screening
[0234] The new PKD1-specific products were generated from one
affected member of each of the 47 Asian families and then used as
template for mutation detection of exons 1-12 and 22-34. Table 2
(above) lists the sequence and PCR condition for primer pairs that
were used for nested amplification of individual exons and their
adjacent intronic sequence. Overlapping pairs were designed for
segments >400 base pairs in length.
[0235] A total of 13 novel variants were detected by SSCA using the
conditions described above. Two are highly likely to be pathogenic
mutations, four are predicted to encode missense substitutions not
found in normals and seven are normal variants (see Table 3).
[0236] The first pathogenic mutation is a G to A transition at
position 9213 in exon 25 that is predicted to result in a nonsense
codon (W3001X). Its presence was confirmed by restriction analysis
using the enzyme Mae I and it was found to segregate with disease.
This variant is predicted to truncate the protein near the carboxyl
end of the Receptor for Egg Jelly (REJ) domain. The W3001X mutation
results in a greatly truncated product missing all of the membrane
spanning elements, intervening loops and carboxy terminus. The
second mutation (T3110C) is predicted to result in a
non-conservative amino acid substitution (W967R) at a critical
position of one of the PKD repeats. The mutation is unique to the
family in which it was found and was not observed in a screen of
over 100 normal Thai chromosomes. The W967R missense mutation is
predicted to disrupt the secondary structure of PKD domain 3. The
WDFGDGS (SEQ ID NO:58) motif within the CC' loop region is the most
conserved sequence of the PKD domains. The tryptophan is replaced
is the first residue of the turn at the end of the C strand and is
conserved in 14 out of 16 PKD domains. Moreover, it is
evolutionarily conserved in mouse and Fugu polycystin-1.
TABLE-US-00004 TABLE 3 Mutations Identified in the PKD1 Gene in a
Thai Population Nucleic Acid Confirmation Patient Exon Change Codon
Change Consequence Enzyme Pathogenic RAMA28-01.sup.0 12 T3110C
W967R Missense (disrupt BsaW 1 (cut NC) PKD domain3) RAMA59-02* 25
G9213A W3001X Nonsense (early Mae I termination) Variants not found
in 100 chromosomes RAMA3-02* 22 T8298G L2696R Missense HinP1 I
RAMA87-01* 25 A9164G R2985G Missense BsrB 1 RAMA87-01* 25 C9326T
R3039C Missense Fau I (cut NC) RAMA45-03* 29 G10064A V3285I
Missense Bsm I Probable normal variants RAMA7-06 2 C474T A88V
Missense Hph I RAMA107-01 2 G487A A92A Silent change TspR I
RAMA94-01 25 C9367T G3052G Silent change Sfo I (cut NC) RAMA66-01
30 A10143G.sup.HG H3311R Missense Nsp I (cut NC) RAMA66-01 30
T10234C.sup.HG L3341L Silent change ASP1 + BseR I RAMA51-01 30
G10255T R3348R Silent change ASP2 + MSC I *Segregation with
disease; .sup.0cannot test for segregation; NC--Normal control;
.sup.HGPresent in one copy of the homologues; ASP--Allele-specific
primer.
[0237] These pathogenic mutations add to previously identified
pathogenic mutations, including a deletion of G3336 (-G3336) in
exon 13, resulting in a frame shift after amino acid 1041 (FS1041);
C4168T (Q1653)X), C6089T (Q1960X) and C6326T (Q2039X) mutations in
exon 15, each resulting in a nonsense termination; -G7205-G7211 in
exon 16, resulting in a FS2331; a C7415T (R2402X) mutation in exon
18, resulting in a nonsense termination; a C7883T (Q2558X) mutation
in exon 19, resulting in a nonsense termination; and a -C8159-T8160
mutation in exon 21, resulting in a FS2649 (Phakdeekitcharoen et
al., supra, 2000). In addition, probable pathogenic mutations
including G3707A (G1166S) and T6078A (V1956E) missense mutations in
exon 15, and a C7433T (R2408C) missense mutation and an insertion
of a GCG trinucleotide between G7535 and G7536 (extra Gly2422) in
exon 18 have been identified (Phakdeekitcharoen et al., supra,
2000).
[0238] Four additional mutations unique to one of the families also
were identified (see Table 3). The mutants segregate with disease,
and were not observed in a screen of over 100 normal Thai
chromosomes. Three of the four variants are predicted to result in
non-conservative amino acid substitutions. Two of them (A9164G,
C9326T) are present in the same allele of a single family (RAMA87).
As such, these mutations meet several criteria expected of
disease-producing mutations, including they are not found in
normal, ethnically matched chromosomes, they segregate with the
disease, and they result in non-conservative substitutions.
[0239] In one case a heteroduplex pattern was discovered for the
exon 22 product of the proband by standard agarose electrophoresis.
The heteroduplex pattern was confirmed to segregate with disease
and subsequently determined that the novel variant was the result
of a T to G transversion at position 8298. This mutation is
predicted to substitute arginine for leucine at position 2696 of
the protein sequence. This non-conservative substitution is within
the REJ domain. Interestingly, the R3039C substitution occurs near
a newly described putative proteolytic cleavage site of
polycystin-1, His(3047)-Leu-Thr-Ala(3050) (SEQ ID NO:59). In the
corresponding position of Fugu and murine polycystin-1, glutamic
acid and arginine, respectively, are present, suggesting a
non-critical role for a non-polar residue at this location.
[0240] Seven nucleotide substitutions that are likely normal
variants were also identified. Two are missense variants that do
not segregate with disease in the family in which they were
discovered. The C474T substitution results in the conservative
replacement of valine by alanine at position 88 in the first
leucine rich (LRR) repeat. The amino acid is not conserved between
species and is not predicted to disrupt the LRR structure. The
second missense variant, A10143G, substitutes arginine for
histidine at position 3311 within the first extracellular loop
between TM2 and TM3. It too, is a conservative change involving a
residue whose identity is not evolutionarily conserved at this
position. The other five variants were silent nucleotide
substitutions that were unique to the pedigree in which they were
found and not found in more than 100 normal chromosomes. It is
possible that these variants can be pathogenic by affecting gene
splicing in the region. Two of the normal variants of exon 30,
A10143G (H3311R) and T10234C (L3341L), were clustered together in a
single PKD1 haplotype. Interestingly, both variants also are
present in at least one of the homologues, suggesting a previous
gene conversion event as the original of these PKD1 variants.
Additional PKD1 variants, which do not appear to be associated with
a PKD1-associated disorder, include two silent mutations, G4885A
(T1558T) and C6058T (S1949S), and a missense mutation, G6195A
(R1995H), in exon 15; a silent T7376C (L2389L) mutation in exon 17;
a silent C7696T (C2495C) mutation in exon 18; and a missense G8021A
(D2604N) mutation in exon 20 (Phakdeekitcharoen et al., supra,
2000).
[0241] Table 4 summarizes the clinical findings for the probands of
17 Thai families. The genotypes and phenotypes for patients with
ADPKD are shown. It has been estimated on the basis of studies of
Caucasian populations that approximately 15% of mutations are
localized to the nonreplicated portion of the PKD1 gene. If the
same frequency is true for the Thai population (the patients were
not screened for mutations in the nonreiterated portion), then the
present studies have identified approximately 45% to 54 percent of
all mutations present in the nonreplicated region. This detection
rate likely can be increased by using more sensitive detection
methods such as DHPLC (Kristensen et al., supra, 2001), HTCSGD
(Leung et al., supra, 2001), or the like.
TABLE-US-00005 TABLE 4 Genotypes and Phenotypes in Thai ADPKD1
Phenotype Renal Genotype insuff. Renal Palpable Liver Heart Valv.
Brain Patients Age Exon Codon Change Consequence HT (Cr >2)
stone kidneys Cyst Abnorm. Aneur Ref. RAMA28-0 30 12 W967R Missense
+ - - - - - - RAMA103- 57 13 FS after 1041 Frameshift + - + - + - -
(*) RAMA49-0 26 15 G1166S Missense + - - + - - - (*) RAMA36-0 47 15
Q1653X Nonsense + - - + - - (*) RAMA108- 57 15 V1956E Missense + +
- - - - (*) RAMA77-0 53 15 Q1960X Nonsense + + - + + - - (*)
RAMA32-0 36 15 Q2039X Nonsense + + - + - - (*) RAMA97-0 45 17
R2402X Nonsense + + - - - - (*) RAMA96-0 30 18 R2408C Missense - -
+ - + - - (*) RAMA99-0 56 18 R2430X Nonsense + + + - + - - (*)
RAMA66-0 39 18 2442 add'l. Gly Extra Glycine + - + - - - - (*)
RAMA55-0 52 19 Q2558X Nonsense - - - + + - - (*) RAMA5-01 53 21 FS
after 2649 Frameshift + + + + + + + (*) RAMA3-02 40 22 L2696R
Missense + + - + + - - RAMA87-0 61 25 R2985G Missense - - - + + - -
25 R3039C Missense - - RAMA59-0 35 25 W3001X Nonsense + + + - - - -
RAMA45-0 59 29 V32851 Missense + + - - - - - HT--hypertension;
Renal insuff.--renal insufficiency; Heart Valv. Abnorm.--heart
valvular abnormalities; Brain Aneur.--brain aneurisyms;
(*)--Phakdeekitcharoen et al., supra, 2000.
[0242] Although the invention has been described with reference to
the above examples, it will be understood that modifications and
variations are encompassed within the spirit and scope of the
invention. Accordingly, the invention is limited only by the
following claims.
Sequence CWU 1
1
113153522DNAHomo sapiens 1tgtaaacttt ttgagacagc atctcaccct
gttccccagg ctggagtgca gtggtgtgat 60catggctcac tgcagcgtca acctcctggg
tctacttgat ctgtaaactt cgagggaagg 120tgtaataaac cctcctgcaa
tgtctttgtt tttcaaaatc tttgtatttc acagtttagc 180ttcgtgggtt
gatgttctat tttgtttttg tgtgtgtgtg tgtgtgtttt gtgttttttt
240ttgagacaca gtcttgctct tgttgcccag gctggagtgc aatggtgtga
tcttggctca 300ctgcaacttc cacctcttgg gttcaagaga ttctcctgcc
tcagccttcc gagtagctag 360gattacaggc gccgccacca caccccgcta
attttgtatt tttagtagag atggggtttc 420tccatattgg tcaggctggt
ctcaaactcc cgacctcagg tgatccgccc acctcagcct 480cccaaaatgc
tgggattaca ggcgtgagtc accgcacctg gccaatgttc tatttttgag
540aacacaacag ttcataatat attctacata gaccatacct gttatgtgta
gataaacaga 600ctcttttccc atttaacacc ttttgcctta ggtttatttt
tctggtatca atactggcac 660acttactttg tttgcagttt cctgtctttt
tttttttttt tttttttttt gagacagagt 720ctcactctgt cacccaggct
ggagtgaagt ggcgggatct cggctcactg caacctctac 780ctcctgggtt
catgcgattc tcctgcctca gcttcccgaa tagctgagac cacaactgtg
840tgccaccatg cccagccaat ttttgtattt ttagtagaca cggggtttca
ccatactggc 900caggatggct caatctcttg acctcgtgat ccacctgcct
ccgcctccca aagtgctggg 960attacaggca tgagccactg tgcctggcct
ttttttttct ttttgagatg gagtctcact 1020ctgtcaccca ggctggagtg
cagtggggta acctcaggtc actgcgacct ccgcctcccg 1080ggttccagtg
attctcctgc ctcagcctcc cgagtagctg ggattacagg cacccaccac
1140catgcctggc taatttttgt atttttagta gagacggggt tttgccacgt
tggccaggtt 1200ggtctcgaac tcttggcctc atgtgacccg cctgccttgg
cctcccaaag tgctgggatt 1260acaggtgtga gccactgtgc ctggcctggc
tttcttgttt cttttctcct cttctagttt 1320ccccctttta ggctaacaat
tattcactgt taataaaaac cctcaggtct gtattttatc 1380aagaaacatt
tccctcacgt cttcttccct gaaccaaaca agatctctgg cacattttat
1440ttgctctgtc tcaccacatg gattttgttt ttttgtttct ttgttttttg
agatggagtc 1500tcactcttgt tgcccaggct ggagtgccat ggcacaatct
cagctcactg caacctccac 1560ctcctgggtt caagcgattc tcctgtctca
gcctcctgag tagctgggat tacaggcgcg 1620tggcaccacc cccagctaat
ttttgtattt ttagtagaga cggggtttca ccatgttggt 1680caggctggtc
tcgaactcct gaccttgtga tctgcccacc ttggcctccc aaagtgctgg
1740gattacaggc atgagccacc acgcccggcc cccatggttt ttcaaatagt
ttagaatttc 1800atttccaggt aactaatttg cttctttaaa catatgtctt
ttctatttaa gaaatccttt 1860ctaaacaatt gcattttatt ccacaaccgc
cttcaaacaa tcattgagac ttggttaatc 1920tgttttgctc atttggcagc
agtttcttgt ggctgtttct tccctccact ggagtccttg 1980aatcttaagt
ctgtcatttg actgcaatta aaagctgggt ttggaataca atcgcagcct
2040taccatccac ctgctgtgtg acctggtaaa tttctttttt tttttttgag
acggagtctt 2100gctctgttgc ccaggctgga gtgcagtggc acaacctctg
cctcccaggt tcaagcgatt 2160ctactgcctc aggctcccta gtagctggga
ttataggtgc ctgccaccat gcccagctga 2220tttttgtatt tttagtagag
atgaggtttc accatgttgg ctaggctggt ctcgaacttc 2280tgatcttgtg
atctgcccgc ctcggcctcc caaagtgctg ggattacagg catgagccac
2340cactcccagc cagttctttt tttctttttt ccattttttt ttttttcgag
acaggatctt 2400actcttttgc ccaggcggga gtgcagtggc acaatcacgg
ctcagcgcag ccactgccta 2460ctgggctcac acgctcctcc ggcctcagcc
tctcgagtac ctgggactac aagcgtgagc 2520cagtttggct aattttggct
aatttttgta gaaacggggt ctcgccatgt tggccaggct 2580ggtctccaac
tcctggactc aagggatcca ccttcctccc cctctcaaag ttctgggatt
2640accggagtga gccactgtgc cctgctggca aatttcttaa actgtctgtg
cctcagtgac 2700ctcatttaat aaagggaata attgtagcac actttttcta
gagctgtgaa gattcaatgg 2760aataaataag gcaataaatg aatggatggg
gaatgaagga tgtgggtttc ctccctcttg 2820tctttcaata agctctcacc
atcaacctcc cattgcctgt tctctctctt ccccctctct 2880ccctctgtct
ctctctcagc caggaaacct ggggtaggga ggcttggagc cagcgggtgc
2940gtcgggaggc tgcgggtact gactcgggcc gcgcacggag atcgcgggag
aaggatccac 3000aaccgcggaa gaaggatcag ggtggagcct gtggctgctg
caggaggagg aacccgccgc 3060ctggcccaca ccacaggaga agggcggagc
agatggcacc ctgcccaccg cttcccgccc 3120acgcacttta gcctgcagcg
gggcggagcg tgaaaaatag ctcgtgctcc tcggccgact 3180ctgcagtgcg
acggcggtgc ttccagacgc tccgccccac gtcgcatgcg ccccgggaac
3240gcgtggggcg gagcttccgg aggccccgcc ctgctgccga ccctgtggag
cggagggtga 3300agcctccgga tgccagtccc tcatcgctgg cccggtcgcg
ctgtggcgaa gggggcggag 3360cctgcacccg ccccgccccc cctcgccccg
tccgccccgc gccgcgcggg gaggaggagg 3420aggagccgcg gcggggcccg
cactgcagcg ccagcgtccg agcgggcggc cgagctcccg 3480gagcggcctg
gccccgagcc ccgagcgggc gtcgctcagc agcaggtcgc ggccgcagcc
3540ccatccagcc cgcgcccgcc atgccgtccg cgggccccgc ctgagctgcg
gcctccgcgc 3600gcgggcgggc ctggggacgg cggggccatg cgcgcgctgc
cctaacgatg ccgcccgccg 3660cgcccgcccg cctggcgctg gccctgggcc
tgggcctgtg gctcggggcg ctggcggggg 3720gccccgggcg cggctgcggg
ccctgcgagc ccccctgcct ctgcggccca gcgcccggcg 3780ccgcctgccg
cgtcaactgc tcgggccgcg ggctgcggac gctcggtccc gcgctgcgca
3840tccccgcgga cgccacagcg ctgtgagtag cgggcccagc ggcacccggg
agaggccgcg 3900ggacgggcgg gcgtgggcgg gttccctggc ccgggacggg
aagcaggacg cgggccagga 3960cgctcccagg ggcgaggctc cggcgcggca
cggcgggccc tgctaaataa ggaacgcctg 4020gagccgcggt tggcacggcc
ccggggagcc gaaaaacccc gggtctggag acagacgtcc 4080cacccggggg
ctctgcagac gccagcgggg gcggggcgcg gaggccgcgc tcagctggga
4140ggacaaacag tcgctaattg gagaggaatt gggatgcggc ctggggctgc
ggggtacccg 4200gagaggtggg gatggctgta gggggcggca gggaagagtt
ccaggaggtg tctggaaaag 4260gatttgatgg atgtgcaaga attgggctga
tgcttaggaa ggggcgatga ggtgggtcca 4320gaagaagggg ggtgaacggt
gtgagcaaag accgtgaggc tggaggctgg ccacgggagg 4380tgtgaggggt
aggggcaggg tgggaggtgg gctcgcgggt gggctggggt catgaagggc
4440ctcaggcgct ctgctattgg gttccaaggc tatcctgaga acaggggtga
ggggggattg 4500ccgtgggggg ttaaagcctt gtcatgttcg ctttcgggag
ataaaaacaa caggtggcct 4560ttatggagac gctgcccaga gccaggtctg
tgccaggctc ctgttggggg tcgtcatgcg 4620gaatcctgac tctgaccatc
cgaggcatag ggaccgtgga gatttgcatt tcacagatga 4680ggaaacaggt
ttggagaggt gacacgacct gtcccaggca tcacagccgg gatgtgcata
4740gcaggggttt ggaactatga ggtgcccagg acccagggtt ggattgaaaa
gggcggaggg 4800gactaagata agcagacagt tgtccccagc gctggggaga
gtcttgggac cagtctgatg 4860ccttgtattt cccaggctcc aggctcctcg
ccgggacagt gtctccttgg gtgcgtgctg 4920gatccctggg ggacgtggca
catccccagg cttgctaaac attgggtggg ttctggcatt 4980tggttttgta
acgtttctgg gtcactcccg cctgtggcca cccttcctta ggggagccgt
5040gtgtccttgg ggctttgctg ggtggtctcg agggtgggag aagaatgggt
tctcctggac 5100caatggagcc cgtgcccctc ggggccacat tgctcctgcg
ctccctgact gcggacgcgt 5160gtgtctcgcg gctgtctctg tggagatggc
ctcctcctgc ctggcaacag cacccacaga 5220attgcatcag acctacccca
cccgttgttt gtgatgctgt agctgagggc tcctctgtct 5280gccaggccgg
tcactgggga ctctgtccag ggcctggtgg ttcctgcttc ccagcacctg
5340atggtgtcca tgagagcagc ccctcaggag ctgtccggga gagaagggcg
ctggtggctg 5400ctgagcggag agcaaggccc gtgttctcca ggcccttggc
acagcagtgg agcccccgcc 5460cctgccttgt gttgtcctct taggctctgg
tcctggggtt tggaggaggg ggaccctggg 5520agttggtggc ctgtcccagc
ctgagctggc aagattccga atgccaggcc ccccaagtgt 5580gcaacagggc
acagggtgac ctcatgtggg caggtgggtg ctgttctgta cacacctggg
5640gccgccgctg ggagagttct ggaaggtggg gtgaggggac ccatggcaaa
ctagggcctt 5700aggaaggatg tgaaggccct ggctggcccc ccaggccacc
ctctgtgctg tggggcagcc 5760cagccatttt gctgtctacc ctgcaaactc
ctcctcgggg agacggctgg gttttcccca 5820gggaagaggg gtcaagctgg
gagaggtgaa ggacacagat cacagctgct ggcaggtgtt 5880caagggtcca
agagcgttgc tgtctgggtg tcaccagtag ccttcctggg gggctcacgc
5940aggtgcctct ccacttgtgg ctccctggct gctgaagctc agcagggaca
gctgtgtcca 6000gttccaggtg gaggacagcc ggggcttctg aggccacagc
ctgccttggg ttaatgatgc 6060tgccgagagg tggtggcttt tggaaaagat
ggcgtactgc aaaacgtgct gctctgcgtg 6120gctcgaagct tcgtggggag
acgtgggcag agccgtggct gactcacaga ccccccaccc 6180cagagcctgc
cctgccctcc ctgccccgac ccttctccct cctgacccat gtgttttttt
6240tttttttttt tttttttgag acagagttca ctcttgttgc caaggctgga
gtgcaatggc 6300acgatctcgg ctcatggcaa cctccgcctc ctgggttcaa
gcgctttttc ctgcctcagc 6360ctcccgagta gctgggatta caggcgtgca
ccaccatgcc tggctaattt tgtattttta 6420gtagagacag ggtttctcca
tattggtcag gctggtcttg aactcctgac ctcagatgat 6480ccgcccgcct
cggcctccca aagtgctggg attacaggca tgagccacca cgcccagccc
6540tgacccatgt tttgaaccaa attccagcca cccttttatc tgcaagcatt
ttggagggca 6600tcgcaatact gcagacccac ctaacacaac agacagttcc
ttcatgccac cgaaggcctg 6660gtgtgttcac atttttggtt taatagtttg
aattaagagc caaataaggt ccacacactg 6720caattagttg atgtcttttt
ttttttcttt tttttttttt ttttgagacg gagtcttgct 6780cttgtctcca
ggccgcagtg cagtggcatg atctcagctc accgcaacct ccgactccct
6840ggttcaagcg attctcctgc ctcagcctcc cgagtacctg gtagctgggt
ttacaggcat 6900gcaccaccgt gcccagctaa tttttgtatt tttagtagag
acggggtttt actgtgttgg 6960ccaggatggt ctcgatctcc tgacctcgtg
atctgcccac ctcggcctcc caaagtgctg 7020ggattacagg cgtgagccac
cgcacccggc caatgtcttt taaaaatata tacttttttt 7080ttttttttga
gacggagttt cgctcttgtt gcccaggctg gagtgcagtg gcgcgatctc
7140acctcacggc aacctccgcc tcccgggttc aagtgattct cctgcctcag
cctctccagt 7200agctgggatt acaggcatgt gccaccatgc ctggctaatt
ttgtattttt aggagagacg 7260gggtttctcc acgttggtca ggctggtctc
aaactcctga cctcaggtga tccgcctgcc 7320ttggcctccc aaagtgttgg
gattacaggt gtgagccaac gcgcccagac aaaaatatat 7380gtgtgtcttt
aaggctggtc aagcaaagca gtaggactgg agaaagaatg aagaattcta
7440cctggctgtg atcaattcgt tgtgaacacc actgtgcttg gaccagctag
ctgatgtctt 7500ttgttttgtt ttgtttgaga cggagtctgg ctctgtcacc
caggctggag gacaatggtg 7560tgatctcggc tcactgcagc ctccatctcc
cgggttcaag cgattctcct gcctcagcct 7620cctgagtagc tgggattaga
ggcgcgcgcc accacgcccg gctaattttt aaaaatattt 7680ttagtagaga
tggggtttca ccatgttggt caggctggtc ttgaactctt ggccttaggt
7740gatctgcttg cctcggcctc ccaaagtgct gggattacag gtgtgagtga
tgtattttat 7800ttatttattt atttatttat ttttattatt tgagatggag
tctcactctg ttgcccaggc 7860tggagtgcag cagtgccatc tcagctcact
gcaagctccg cctcctgggt tcacgccatt 7920ctcctgcctc agcctcctga
gtagcctgga ctggtgcccg ccaccatgcc cagctaattt 7980tttgtatttt
tagtagagac ggggtttcac cgtgttagcc aggatggtct ggatctcctg
8040acctcgtgat cctcccgcct cagcctccca aagtgctggg attacaggct
tgagccaccg 8100cctgtctttt aaatgtccga tgatgtctag gagcttccct
tcctctcttt ttccttgtgc 8160aatttgttga agaaactggc tcctgcagcc
tggatttctc gctgtgtctt gggggtgcca 8220cctccatggt gtcacctccg
tggtgctgtg agtgtgtgct ttgtgtttct tgtaaattgg 8280tcgttggagc
cgacatccca ttgtcccaga ggttgtcctg gctggcactg gcctaggtgt
8340agatgtcatc agctcagggc cccctgctct aaaggccact tctggtgctg
gttgccactc 8400accctggctg ggggtcacct gggtctgctg ctgtctcgca
aatgctgggg tccaggactg 8460ggcacatcga gggacttggt aggtgcttgg
ttcactgatg taaaatatag gagcacccgg 8520ggccttgccc tttcccacct
gcatccctga atgacaggag agtgtgggag agtgtaggga 8580cagcaggcgc
agaccccggg gcccctgcct gggattggcg tcggggaaga caggcattct
8640ggagcgaccc ctaggcctga tgccttagag cgcaactgcc agagacacag
cttccttggg 8700gggctggcca ggccacggag gggccctggc tcccatttct
ggtccctgga tcctgagagc 8760gaggactagg gattgtcacc aaggcctcca
tgagccctca gcagaaggag ggccaccctc 8820gagggctccg ttatcactgg
agcccgcgtt caaccaacac gcagatgatt ctccaaggac 8880agagatggat
gatggggagg gggctggcct ggaaggaccc ccagtgcagg tgacattgaa
8940gccaggtttc aaagctccca cagggagctg cccagagaga gtccccaagg
ggcaaggtga 9000ctcgggggca ggggtagggc ctctgtcagg agagcctagg
agaggcctgt gtcttctagg 9060aagagccctg gcagccgagc ggaggcagtg
gtgaggacct gcatcctgca tgtccagctg 9120gcctcacccg gggtccctga
gccgggtctt acgtggctcc cgcactcggg cgttcagaac 9180gtgcctgcgt
gagaaacggt agtttcttta ttagacgcgg atgcaaactc gccaaacttg
9240tggacaaaaa tgtggacaag aagtcacacg ctcactcctg tacgcgattg
ccggcagggg 9300tgggggaagg gatggggagg ctttggttgt gtctgcagca
gttgggaatg tggggcaccc 9360gagctcccac tgcagaggcg actgtggaga
cagagagcac ctgcaggtca tccatgcagt 9420atcggcttgc atccagatca
tacagggaac actatgattc aacaacagac agggaccccg 9480tttaaacatg
gacaaggggt cactcacgcc tggaatccca gcagtttggg aggccagggt
9540gggtggatcg cttgagccca ggagtttgac accagcctgg gcaacagggt
gagaccccgg 9600tctctaaaaa ataaaagaac attggccggg cgtggtggta
tgcatctgtg gtcccagcta 9660ttcaggagac tgaggtggga catcacttga
gccgaggagg tcaaggctgc agtgagctgt 9720gatcacacca ctgcactcca
ggctgggtca cagagcaaga ccctgtctca aaaaaaaaaa 9780aaaaaaaaaa
aaaaaatcac aggatctgaa cagagatttc tccaaagaag acgcacagat
9840ggccaacagc gtgtgagaag atggtcggcc tcattagtca tgagggaaac
gtaaatcaaa 9900accactgtcc agccgggcgc ggtgcctcac gcctgtaatc
ccagcacttt aggagagcag 9960atggcttgag gccaggagtt tgaggccagc
ctgggcaaca tagcgagacc aataaataga 10020tattagtggt ggcgcctgta
gtcccagcta gttgggaggc tgagggggga ggattccctg 10080agtctatgag
gttgagactg cagttagctg tgatggtgcc actgcactcc agcctgggcg
10140actaggaaac ggtctttaaa aaaaaaaaaa aaaaacaggg tgggcgcggt
ggttcacgcc 10200tgtaatctca gcactttggg aggccaaggt ggggggatca
caaggtcagg agtttgtgac 10260cagcctgacc aacatggtga aaccccgttc
tactaaaaat acaaaaatta gcgaggtgtg 10320gtcgtgggcg cctgtaatcc
cagctaatta ggaggctgag gcaggagaat cacttgaacc 10380cgggaggcgg
aggttgcagt gagccaatat cacaccactg cactctagcc tggtcaacag
10440agcgagactc tgtctcaaaa aaaaaaaatg ctgagcgtgg tggcgcatgc
ctgtagtctc 10500agctactttg ggggctgagg caggagaatc gcttgaacct
gggaggcaga ggtcgcagtg 10560aggcaagatt gcaccattgc actccagcct
gggagacaga gtgaaactct gtctcaaaaa 10620gaaaaggtct aggaagagtc
cgcaccctct ccccgcggtg gccacgccgg gctccgcgct 10680gagccctctg
tgttcttgtc tctccatacc tcatcacggc accgcagggt tgcagccact
10740cctggtctca ttttacacac caggaaattg aggctctttg agaagccgtg
gtgatgattt 10800catcagcatg ctctggggca gacccctgca gccgcacagg
gtgcctgggg cccacactag 10860tgccctggtt tatagacaga cagaggtggc
agtggcgctt ccgagtcggg ctgcgatgtg 10920cttgcactcc ccgaggggct
gaggggccct gcgcccaggt gcagctgctt gggtgctgcc 10980agcccctccc
acctctccct ccctgccagc ccctcccacc tctccctccc tgccagcccc
11040tcccacctct ccctccctgc cagcccctcc cacctctccc tccctgccag
cccctcccac 11100ctctccctcc ctgccagccc ctcccacctc tccctccctg
ccagcccctc ccacctctcc 11160ctccctgcca gcccctccca cctctccctc
cctccagccc ctcccacctc tccctccctg 11220ccagcccctc ccacctctcc
ctccctgcca gcccctccca cctctccctc cctgccagcc 11280cctcccacct
ctccctccct gccagcccct cccacctctc cctccctgcc agcccctccc
11340acctctccct ccctgccagc ccctcccacc tctccctccc tggctcatcc
ctgctgtgtc 11400ccttctctct agtttcctgt tcagtttcag gaaggaggct
gggaacccag atgtagggaa 11460tttgcgccct ggagtcagac ctgggttcac
gtcccagcgc ctccacctct ggtgtgacct 11520tggtccagtc tctcagcctc
agtttcctca cctgtaaagt gggctccatg attagatgca 11580ccctgcaggg
cagtgtagca gtgacctggc tcagccactg gcagccccaa caatcatacc
11640ttgttaaagt agctctgtcg gttccctcag gggttccggg ggcccattcc
cctgtcctcc 11700atgcactgtg agacctgccc tgccacagag cagagtgtaa
cagcctgagg gtgagagcca 11760gacactgtgc ctgtgcttag accagacact
ggacgacggg agccagtgca gcctgggcgg 11820gtggactcct atggacccct
cagcacccag cctcggtgcc ttcagcgcag ggccgcgtgg 11880ctgtgggggc
tcacaagacc cggcccactc ctgcttgtgc ctacatctgg gtgtttgccc
11940attggtgcct tttgacgcgt tctggtgtgt gtgagacgtg cggggctggg
aagtgttggc 12000agagccgcga gtaccgtcct cactcctttt gttcttttga
cgtaagctgg cgagtggcac 12060tgcctgagtt ccgctcagtg cccgccctga
tgtgcggacc ccgctgcatt cttgctgtta 12120ggtggtggcg gtgtgcgctg
tcgctggtgg gcaccgagag tctttgggag ctttggggag 12180gttgtgccaa
gcctgagcct cgacgtcccc cttcccggct ttctgttggc tcttctgagg
12240ccagggcatc tctatgaggg cctcctgctg gagccgtctc tgtggatctc
ctctgccatc 12300ctggcccatg agtgggtgat gcgctggcca ccatctggtg
acagtggccg ggcaccgctg 12360ccaaatgtgg gtcccgcatc tgcaagcccc
tccctgggtc ccctagggta tggggtggtt 12420ctgccactgc cctcgctccc
ccaccttggg gtgcctctcc ccctgctcgt gggggagacc 12480ctgcctggga
tctgctttcc agcaaggaat atactttgga gggagacaca catgttcttt
12540tctggagctc tgcagtggcc acggcagccc agcccgccaa gcaccctgga
atgaaaacat 12600cccgctgctg tctgggcctg gcctgcactc tgctgcctgc
gctccagctg gctgaggccg 12660ggcacgtctg cgggcacagc agcgggggcg
ccacagtctc cctgcagagt gagcgcagct 12720ggaaaatgca gctcacgccc
tttcccagaa cacctcgctc ttcatggctt ggcagctgtc 12780cttgcctagg
ggccagggtg cccaggcact ggtggcagga gaagggctac atctggggct
12840gaggcgggct gggtcctttt ctccctgcag ctcccgaggc ccagccctgg
cccagcctgg 12900cattcctgac cttagcagcg ccatgatctg aagacaggct
ggcttctgtg aggccacctc 12960agaaagggct ttgtgcccag gcagaggcgg
aagccagctc ttccttctgg ttgaggcagg 13020aatgaggcca gcgctgggca
agcccatgcc cagggaacgt cacagctgtg ggagtacagg 13080ggctccgggt
tctgagcccg tccactgtgc atcgtggccc tggcctcagg atggctcgta
13140ccatcattgg ctgtgcccac agccgagtgg gtgatgggat tccggctgcc
ccgctggatc 13200tgtgctgctg ccctctccag ggcactgctg tgcccgcaca
gccgggcgca gatggccagt 13260ttgcttgccc ccccccccac catcctcttc
ctaccttggc ttcctccatt gacacactgg 13320accctgctgg ctgcccgggg
aggtgtttgg gggatggtgt tgggggagga ggagggcccc 13380ttgagcctca
gtgtgcccat caggagcgta aggtcagtgc agcacctgcc cacacaggct
13440gtgaagggtg ggagtggaga gggatgcaag ggggtcacaa cgcctggctc
catgtcagct 13500gcgtgcaggg gcaccaggag ccggccctca ttctcccctt
gaactggaag ggtggccccg 13560accccagcgg caggtagcat acgtatgaag
cgctctcctt cctacacccc acaggtgggc 13620tcgtctccag acggcccttt
ttgagctggc tgtgtttttc catctgtgta ggcaaggaca 13680tcgcagactc
ccctttctca tctccctcgt tcagcctccg aggccggagt ctccatccct
13740gtgcctgcct gtgggtcccg ggaggacctg aggctgccca tgtcaccccc
ggcatctcat 13800cctggggaca gttcagccgt gggagggatc tgtaaggaca
gaatgccgct gagcctgggg 13860ctccccagct agtctcacac cccgtgtctg
ggacccagag accctcgtgc agggctctgt 13920tgcttggggc ctggcagcct
cgtcctgtat cagaggctgc cacccccacc cctcgtgggg 13980ccagggttgt
ggccggcctc cctggccctc cccatggaag tggtaggcgg agccagcagc
14040catctgccca gcccggggct gcactgtttt ttttcaaatg agcaccgtcc
caaactgcag 14100cccgttaatt taaacaggat catttccggc cctggaagcc
gcctcactct ccttaaatag 14160aaaggagcac agcgcagagg gaaacagatg
aggtcatggc tcggctggcc cagcgaggaa 14220ggggccgcag tgggggtggc
actgccgcct gtcccctgtc ctctccagcg cccacactgc 14280agcccatttc
ctcaccctgg gcctgctctc gggagggacg ggcctggggg tcctcttgct
14340gggcggaggg gaaccagctc ctccaggaga ggacggggcc tggcaggggg
catggggcct 14400ccctgggtct ggcgtcctgt cctgcccctg ccgagggagg
agcggttaca taagctccgc 14460aggcggcccc tccgagccgg tccccccagc
ccagtttcca gtgaggcggc cagcgcgggc 14520gggggtgccg ggcctggcgc
acacccgctg ctgaccacac gtgtctggaa tgtgcagatg 14580tttctttggg
ggctccgtcc ggcccccaga ccccactcag catctggtct ggggagtggg
14640cgcctggggc actcagctct gagtgtgaga ctctgaggca ggtctggttt
gtctggggcc 14700attccctctg ctgtggattg ggagggcccc gggagctgcc
ccacacccag ggaagttctc 14760ctcagtccca ctgttgcatt ccccgacccc
ggctcccccg gcccaggagc gcctgtgggg 14820cagaaggccc agccccaaga
cttcccggcc ctgccagcct caggcttcac ccaccctcgc 14880gccaactgtg
ggcagagccc agggggaggg caggagagcc agcgcctggc tgggaacacc
14940cctgaggggc cgaggctcca gggcgagggg gcccgacctg gggttcacac
gcccgggtgg 15000cgggcagacc cgctgcagca tgagacacgt gtcagctacc
tcgggccggc aggctggccc 15060tgctgcccac agccctggga cgtggcccca
cctgtgacgg gtgtggaggg gcagcctcca 15120ggcctggcca caccctctgc
tgttgctgct cctgctccag gattggcaag ggtgctggga 15180aggggtgaag
acccgtactg tggccacaca cctgggactt ccttctccac ccagtggtgc
15240cccagcagcc gctaaggagc ccgctgggtc ccacgctagg atggtcctaa
ctcctcccgc 15300cttccagatc ggacgctcgg cgctggggac cccttgtgtc
ccggggctgg ggcaccgtcc 15360tgcccccatg ggggtgtact cctcccgaca
agcttggctt cagcttccct gggagcacat 15420cctggccctc gggcacccat
caggctgtcc ctgtgcacct ggctcccacc cttccagctc 15480atagcaggaa
ctggggtgag gagtgcgtgg ggcagcaagg gcctgggacc ccagaggacc
15540ctgcactctg ctctgtgctc ttgcctgggc ttagggccgc tcggtggtcc
tgctgccaga 15600tgcctgggcc ctgctgtgtc ccccatcctt gcagggaacc
agaacgtggg ggcagggcat 15660cagacagcgg cgatgatgtc acctggcggg
tgcagaggaa gcccgagggg cggggtgggg 15720gggctggcgc gaggctgcct
ggctaggcct tggcgttccc ccagaacggc gatggcaaaa 15780gcagatggag
acgtgaaaaa gtacgggagc aagcgaggtg aggactccac ggggacccct
15840gtgctgttcc ctgtccctga agcccacacc tgagtcctgc ccagggcaga
tgcttccaca 15900cccagggggc acctgagtcc tacccagggc agacgcttcc
acaccctggg ggctggggga 15960ctgcacctgg ctcctgtctg ggccccagct
tcattccact gccctgggcc ctgggagctc 16020ggccgagcgg ggtccccaag
accttgctgc atttctgggc cttgggctgg ggtgagggcc 16080gggagaagga
gccagcctgg agcctggcac gcagggagtg catggccaga accggtgaca
16140ggcagggctg cctgctggcg tggaagaagt gtccatggca cccccaggcc
tggttcacag 16200tgggatgggc ggggagccgg ggggctctgg ggtcctcggc
tgacctgccc ccacccctgc 16260cctggcttgt cagctcccag cagcagccac
tcttgatgga ttttccagaa aatgaggtgt 16320ggccaaacat cttcaggctt
ttccttcttt cctttctccc gtggcctggg tgggagctgc 16380tccccatgcc
tgggggcagg tgcgagagcc tgtgcccctc cctggggcag tttcacagct
16440gtgtcccttc cagggggcct gcctgtgttc accgtggcct ctgcagcacc
tctcgcccct 16500tagggctcct gcgcctcggg tcccggtgcc tcatttctcc
ctaaagcatt ggttctgctg 16560ccgccgcagc cgctggaaag tccctcctca
ggtctaactg cagttcctca cggcacagtg 16620ttccccctcg ggcatggtgc
ttgggcagtg ggtgtgagtc cagctgcctc accctgtctc 16680gagaatggcc
tcttgctggt ctcccagcca ccaccctgtc ccaccccacg gcggggatgg
16740tgtggatgcc tagcagcgcg gctgtgggcc cacccatcct tatgggcagt
ggggagcacc 16800tcagcccgtg tccctacctt ggtgtagagg aggggacggc
agagaagcag ggttcagtta 16860ggggggaagt ggtggccctg ccggaggggc
cgttccctgt gtgcctggcc cccagatcct 16920ctcccctccc ggagcccagg
gcacaggcat aggctctctg agtgtcccac agcccctggg 16980ggaagggaac
tgcaccccca accgtgccct ccatccgcag atggaacgag aagctccggg
17040agccagtgcc cagcgtctca tctgtctggg cacccagccc aggtgagggc
ctggctccac 17100cgtccgtggc tggtgctgct tcctggcacg gagaaggcct
cggctgctct gtcccctcag 17160ctggggtggc ctctggtccc cttctttgtt
ggttcccttc tcaagctctt gccctggccc 17220cgggccccac cgggcagcct
gtgtgtgcgt ctctcctgcg ccgggtaggc tcctgtggga 17280gcggagctcc
ggtgggagga gcagggctgg aggctggcag gggctgggcg ggtgttcagg
17340gatggaggcc gccccggctt ggggctggct gccgggtggt cattgctggg
aagagcaagt 17400ctaggcggag gcacctgctg ggtcactcgt ggggagggtg
acacctgggg aagtagaggc 17460ccgtggcagg aggtgaggcc tcggggtcct
ggggagcagg ggggtggtgt gcagacctgc 17520ggagccatag tcctgtgcca
ggagcactac tgggagtgcg tgggaccagg aggggtgccc 17580agggtgggcg
gcagagtgac ccccgaggtg cttgaggccg aggggaggtg gagttctcgg
17640tttgccccag ctctctgtct actcacctcc gcatcaccag ctccaggacc
tggtttgtaa 17700ctcgggcagc tctgaaaaga gagacatgct gccgccctgt
ggtttctgtt gctttttctt 17760cactgactac tgacatggga tgtttttcct
acggctgtga ccaattgtgc ttcttctaat 17820tgcctggttt ttcttttttt
gtttttggag ttttctcttt ctttcctccc tccctctcac 17880cctccatcct
tttttttttt atttttattt tttgagatgg agcttcactc ttgcaggatg
17940gggtgctgga gtgcaggggt gcgatctcag ctcactgcaa cctctgcctc
gcgggttcaa 18000gtgattctcc tgcctaagcc tcctgagtag ctggaattac
aggtgcttgc caccacgccc 18060gactaattct gtagttttgg tagagacagg
gtgtctccgt gttggtcggt ctggtcttga 18120actcctgacc tcaggtgatg
cgcccgcctc agcctcccaa agtgctggga ttacaggcag 18180gagccattgc
acccggctct ttccccttct ccttttcttc tctctctcct ccctttcttt
18240cttttctttt cttttttttt tcttttgaga tggagtctcg ctctgtcacc
aggctggatt 18300gcagtggcgt gatcttggct cactgcaacc ttcgcctccc
gggttcacgt gattctcctg 18360cctcagcctc ctgagtggct ggcactacag
gctcccgccg ccatgcccgg ctaatttttg 18420catttttagt agagacaggg
tttcaccctg ttggccagga tggtctcgat ctcttgatct 18480catgatccac
ccaccttggc ctcccaaagt tctggcatta caggagtgag ccaccgtgcc
18540cggccatctt tctttccttg ctttctcttt gttttctttc gagaccgggt
cttgctctgt 18600cgcccaggct ggactgcagt ggcacaatca tagctcactg
cagcctcgac ttccctggct 18660caagcgatcc ttcctcctca gccccccgag
tagctggaac tacagttaca cactaccatg 18720cctggctgat tctttttttc
cttgtagaga tggggtcttg ctatgctgtc catcctggtc 18780tcaaactcct
ggccttccca aagcactggg tttacaggca taagccacca cacccagttt
18840ccttttcttc tttttaactg gaatagttga cgttttcttt attagctgtg
tgtcaggagg 18900gtatttttgg cctttagtat gtcgtgtaag ttgctagtgc
ttttctgaga ttgtagtttg 18960ttttctaatt ttatttatat tttgcgtaga
agttgtgtat tttagatgga gttaggtcgg 19020ctggtctttg atgttttatt
tattaattat gtatgtattt atttattttt gaggtagagt 19080ctcgccgttt
cacccaggct ggagtacagt gatgcgatct cagctccctg tagccttgac
19140ctctctgggc tcaagtgatt tttctctcct ctacctcccg agtacttggg
accccaggcg 19200catgccgcca tgcctggcta atgtgtattt tttgtagata
cggggtctca ctgtgttgcc 19260cagggtggtt tcaaaatcct gggcccaggc
gatccttccg tctcagctcc cacggtgctg 19320tgttaccggc gtgtgcccag
tgcctggccg tcttggaggt cttgtttctc tgggtttatg 19380cctcgaggtg
gcgcctgctc ccctgtgctc cctggtagcc tggtagtgag cctgcttctc
19440acacagtcat acctggttgt ggtcccacag tgggaccacc ctgttgggtt
cagaacagga 19500gatgggggcc cctcgagtct gtgtgggggc tgtggacagg
gttgggagac cttggctctg 19560tgggggactg tggacagggg atggggggcc
ttggccctgc gtgggatggg ttgggggtcc 19620gtgcccttcc tggccctggg
tggacaggtc catgtggcac tcggcatagg gctgagatgg 19680gtgcagaggg
ctgaggcccc caggcctctc ctggcttggt ttccccagat gagtgttcat
19740ttgggtcttc catcagaaag tcccctcctg acctctggga gtggggagct
caagggtggg 19800aggccatagc ttggggatgc tggcaatgtg tgggatgggc
ccagggaagg cctctggcct 19860actaggggct ctggccctga cccacggcca
ctcactcctc agagacgtct cccacaacct 19920gctccgggcg ctggacgttg
ggctcctggc gaacctctcg gcgctggcag agctgtgagt 19980gtcccccagt
cgtgccagca tgcggggctc actccgggtg ggctggcggc accgcctctt
20040gctgctcagc tgtgggggct tccatcagct ttgccgaatc ccccgtctct
tccagggata 20100taagcaacaa caagatttct acgttagaag aaggaatatt
tgctaattta tttaatttaa 20160gtgaaatgta agttgtggtt ctttgggtgg
ggtcctggct ggaccccagg cccccaatat 20220cccttctgcc ctcccagttg
gtccgtgtcc ccttccaggc ttgagaccag atcctggggg 20280cagttcactg
cctgcttgga gccccccagt gccggcttgg ttggggcagg ggaggcggtg
20340ctgtcagggt ggctccaggg cctggttgcc agtggggggc tggcatagac
ccttcccacc 20400agacctggtc cccaacacct gcccctgccc tgcagaaacc
tgagtgggaa cccgtttgag 20460tgtgactgtg gcctggcgtg gctgccgcga
tgggcggagg agcagcaggt gcgggtggtg 20520cagcccgagg cagccacgtg
tgctgggcct ggctccctgg ctggccagcc tctgcttggc 20580atccccttgc
tggacagtgg ctgtggtgag tgccggtggg tggggccagc tctgtccttc
20640ccagccaggt gggacctggg ccctgcagac actgggcagg gctcaggaag
gcctctctgg 20700ggggggcctc cgggccaagg gaacagcatg ggagcctgtg
agtgcggcgg gcggatgtgg 20760gggcgtgggg tggagccagg aggagcagaa
cccggggtcc agtggctgcc tcttctaggt 20820gaggagtatg tcgcctgcct
ccctgacaac agctcaggca ccgtggcagc agtgtccttt 20880tcagctgccc
acgaaggcct gcttcagcca gaggcctgca gcgccttctg cttctccacc
20940ggccagggcc tcgcagccct ctcggagcag ggctggtgcc tgtgtggggc
ggcccagccc 21000tccagtgcct cctttgcctg cctgtccctc tgctccggcc
ccccgccacc tcctgccccc 21060acctgtaggg gccccaccct cctccagcac
gtcttccctg cctccccagg ggccaccctg 21120gtggggcccc acggacctct
ggcctctggc cagctagcag ccttccacat cgctgccccg 21180ctccctgtca
ctgccacacg ctgggacttc ggagacggct ccgccgaggt ggatgccgct
21240gggccggctg cctcgcatcg ctatgtgctg cctgggcgct atcacgtgac
ggccgtgctg 21300gccctggggg ccggctcagc cctgctgggg acagacgtgc
aggtggaagc ggcacctgcc 21360gccctggagc tcgtgtgccc gtcctcggtg
cagagtgacg agagcctcga cctcagcatc 21420cagaaccgcg gtggttcagg
cctggaggcc gcctacagca tcgtggccct gggcgaggag 21480ccggcccgag
gtgagtgtct gctgcccact ccccttcctc cccagggcca tccagatggg
21540gcagagcctg gtacccccgt cttgggccca cactgaccgt tgacaccctc
gttcccaccg 21600gtctccagcg gtgcacccgc tctgcccctc ggacacggag
atcttccctg gcaacgggca 21660ctgctaccgc ctggtggtgg agaaggcggc
ctggctgcag gcgcaggagc agtgtcaggc 21720ctgggccggg gccgccctgg
caatggtgga cagtcccgcc gtgcagcgct tcctggtctc 21780ccgggtcacc
aggtgcctgc ccccaccccc cgaggggcca taggttggga gatctctgaa
21840gcactggggc agagactgcg gctggggagt ctcaggagga aggaggtggg
agctgggccg 21900gccctggtga gcaggtggcg ccggccggtg gggccgttcc
tgtcagctct gcagatgcag 21960aggtggacat gagctggggg cagcctccgg
acactcctgg gcacgccata cgggaggtgg 22020cctgcacggg gatccctgcc
ggtacccaca ggccccgtgg gtgggtgctg ctgtgagcct 22080gggctggtgg
gccctggtct ccgggctctg agcctcagtt tccccatctg gaaaggggga
22140cagtgatggg gctcccagcg ggctgctgtg agggtgggag gatggaggag
tgccctgagc 22200cccctgccat cccacacccg cccccaggag cctagacgtg
tggatcggct tctcgactgt 22260gcagggggtg gaggtgggcc cagcgccgca
gggcgaggcc ttcagcctgg agagctgcca 22320gaactggctg cccggggagc
cacacccagc cacagccgag cactgcgtcc ggctcgggcc 22380caccgggtgg
tgtaacaccg acctgtgctc agcgccgcac agctacgtct gcgagctgca
22440gcccggaggt gtgcgggggg ccaggcaggg gcctgagacg ctggctgtgg
ttaggggcct 22500gccgagcgcc cgcggtggag cctgggctga ggaggagggg
ctggtggggg ggttttcggg 22560cggctcggtc cccagtctgt tcgtcctggt
gtcctgggcc ctggcccggc gcctcactgt 22620gcactcgcca ccccaggccc
agtgcaggat gccgagaacc tcctcgtggg agcgcccagt 22680ggggacctgc
agggacccct gacgcctctg gcacagcagg acggcctctc agccccgcac
22740gagcccgtgg aggtagtcgg ccccccacgt tctacaacct gccctcctgc
ctgcccctgg 22800aggccttgcc tgccctgccc actgtgggtc tcgccaaaaa
acttgggggc cttaatgttg 22860cttgtgccca gtgaagatgg ttgggaaaat
ccagagtgca gagaggaaag cgtttactca 22920cattacctcc aggccttttc
tctgagcgtg tgtgagttat tcctgaaagg caggtcaggg 22980gtcctgcccc
ccatggacag tttccaccgg agtcttcctc tcgagcgaca ggagccaggc
23040ctgtgggggt ctgatggctc gctctccttc cctcccctct tcctgggaag
ttcgggtagg 23100gggagtctgg gcttcaggct gggatggggt ctgtggagct
gaggcggccc cctgcccacc 23160aggtcatggt attcccgggc ctgcgtctga
gccgtgaagc cttcctcacc acggccgaat 23220ttgggaccca ggagctccgg
cggcccgccc agctgcggct gcaggtgtac cggctcctca 23280gcacagcagg
tgggactctg ggtggtgggt ggtgggtggt gggcgccgca ggactcgggg
23340tggcctctct gagctttcac gtctgctggt cctgtggcca ccagagtggt
tcccagtctt 23400aggtggacag agcaggggtt ccagagacac cagctcattc
caggtgtcct gggggtggat 23460tgggtggggc ctgcctgggg gccggcctgg
gtcagtcggc tggccggaga cggacgcagc 23520actgggctgg gagtgctgcc
caggtgggga gacctgtcct cacagcaagg ccaggattgc 23580tggtgcaggc
agttgggcat ctctgacggt ggcctgtggg caaatcaggg ccccaacacc
23640ctcccctcct cacagggacc ccggagaacg gcagcgagcc tgagagcagg
tccccggaca 23700acaggaccca gctggccccc gcgtgcatgc cagggggacg
ctggtgccct ggagccaaca 23760tctgcttgcc gctggacgcc tcctgccacc
cccaggcctg cgccaatggc tgcacgtcag 23820ggccagggct acccggggcc
ccctatgcgc tatggagaga gttcctcttc tccgttcccg 23880cggggccccc
cgcgcagtac tcggtgtgtg gccctgacct gggtctgttc cctgcatctc
23940ctcaggccac cttcctgtct gctgcccagg gtctgggtct gtgcaccaga
cacacccagc 24000ctgcaggccc ctcccacgtc cttgccacct ctgacctccg
acctctgcag tgccctcggc 24060cctctcccag tgggagaagc tctcgcctgg
gcccttggca cgagctgtgc ctcctcttcc 24120tctctcccag cacagctgct
ccttcctgtc tgccaggtct tggcctgtgt cctctccccg 24180tgtgtccccc
ggtctgcaac tgtcctgcct gtccttgtca cgagcactgt ggggaggctc
24240cttgaggtgt ggctgacgaa gcggggagcc ctgcgtgtcc accctcatcc
gtcgtgcggg 24300ggtccacggg ccatgaccgt gaggacgtga tgcagccctg
cctccctctc cacaggtcac 24360cctccacggc caggatgtcc tcatgctccc
tggtgacctc gttggcttgc agcacgacgc 24420tggccctggc gccctcctgc
actgctcgcc ggctcccggc caccctggtc cccgggcccc 24480gtacctctcc
gccaacgcct cgtcatggct gccccacttg ccagcccagc tggagggcac
24540ttgggcctgc cctgcctgtg ccctgcggct gcttgcagcc acggaacagc
tcaccgtgct 24600gctgggcttg aggcccaacc ctggactgcg gctgcctggg
cgctatgagg tccgggcaga 24660ggtgggcaat ggcgtgtcca ggcacaacct
ctcctgcagc tttgacgtgg tctccccagt 24720ggctgggctg cgggtcatct
accctgcccc ccgcgacggc cgcctctacg tgcccaccaa 24780cggctcagcc
ttggtgctcc aggtggactc tggtgccaac gccacggcca cggctcgctg
24840gcctgggggc agtgtcagcg cccgctttga gaatgtctgc cctgccctgg
tggccacctt 24900cgtgcccggc tgcccctggg agaccaacga taccctgttc
tcagtggtag cactgccgtg 24960gctcagtgag ggggagcacg tggtggacgt
ggtggtggaa aacagcgcca gccgggccaa 25020cctcagcctg cgggtgacgg
cggaggagcc catctgtggc ctccgcgcca cgcccagccc 25080cgaggcccgt
gtactgcagg gagtcctagt ggtgagtatg gccgaggctc caccaccagc
25140ccccaggcag gtgcctgcag acagggtgct cacacagggc gtgaggcctg
gcttcccagt 25200gagggcagca gcccagttac tggggacgtc ggccccgggc
aggtcctgct ggctggctcc 25260tcgggctacc tggtgggctt taaattcctg
gaaagtcacg gctctgacag tggctccgct 25320aactcattcc actgtctcat
ttcacaaaat gaatttaaaa ctctgctccc tgacctcaca 25380cgagcccccg
tgagtctctc acgccctctg ctgtgttctc gcctggctaa agcgagtggc
25440ttttgaggtg gagtctgaac ccctgatggg aaactgcggg ctgcccgcgg
tgccaccatg 25500ctgggtacat gggggacagg gctgtctcca tcttgcgggt
acctgcctct tcaccagggg 25560ccttgggagg ggccatcaga aatggcgtga
cctgtgcagc ctgtcctggg ttctgtaagc 25620cagtgtaggt gcctcccctc
actgctccga gctctctggg tgaggagctg gggcaagagc 25680gccgggaggg
tctgagaaga ctcagagaga ggtggactct ttgtagctgg tactaggttt
25740gctttacaga tggggaaact gaggcacaga gaggttgagg cattagtagt
actacatggc 25800tggctggaga gccggacagt gagtgtccca gcccgggctt
ggctcccatg gcatgcagag 25860ccccgggcac ctcctctcct ctgtgccccg
cgtgggactc tccagcccga cgggaggtgt 25920gtccaggagg cgacaggcta
agggcagagt cctccacaga gcccaggctg acaccattcc 25980ccccgcagag
gtacagcccc gtggtggagg ccggctcgga catggtcttc cggtggacca
26040tcaacgacaa gcagtccctg accttccaga acgtggtctt caatgtcatt
tatcagagcg 26100cggcggtctt caagctctca gtaggtgggc gggggtgggg
aggggagggg atggggcggg 26160gcagggcggg ggcgggctcc accttcacct
ctgccttctg ctctgcttca tgctgcccga 26220ggacgctgcc atggctgtgg
gtgagtggag ggagggacgc caatcagggc caggcctctc 26280acctgccacc
tgggctcact gacgcctgtc cctgcagctg acggcctcca accacgtgag
26340caacgtcacc gtgaactaca acgtaaccgt ggagcggatg aacaggatgc
agggtctgca 26400ggtctccaca gtgccggccg tgctgtcccc caatgccacg
ctagcactga cggcgggcgt 26460gctggtggac tcggccgtgg aggtggcctt
cctgtgagtg actcgggggc cggtttgggg 26520tgggcaccag gctcttgtcc
cagccccagc ctcagccgag ggacccccac atcacggggt 26580tgcttttctg
agcctcggtt tccctgtctg ttgggaggta actgggtgca caggagccct
26640gaggctgcac gggagccggg agaggcctca gcacagccgg gtgggccctg
aatggaggcc 26700cggggcgtga ctgcagagtg gagcctcggc tgggtcccaa
gcaccccctg ccccgccacc 26760gcccacccct gtcccggttc actcactgcg
tcccaccgcc ccggcaggtg gacctttggg 26820gatggggagc aggccctcca
ccagttccag cctccgtaca acgagtcctt cccggttcca 26880gacccctcgg
tggcccaggt gctggtggag cacaatgtca tgcacaccta cgctgcccca
26940ggtgagggat gagggggtga gggggccact gcctttcagg ctctgagcac
gggtcccccc 27000agctccccag tcaagctgcc ccccttcctc cccaacagcc
ctcactgtga cctcacctgg 27060gctgatggct taggccctac tggggtgagg
gaggggccag gcgtgggggg agtggacagg 27120gaagctgggc ccctgaactg
cgccccccgc cctccccggg cctggctctt gctgctctgc 27180tgccccgagt
gcagctgcac ttggaggcgg tgcgtcctcg ccaggcagcc ctcagtgctg
27240ctacacctgt gctccgtccc gcacgtggct tgggagcctg ggacccttaa
ggctgggccg 27300caggtgcagc cgttcacccc gggctcctca ggcggggggc
ttctgccgag cgggtgggga 27360gcaggtgggg gtgccgcggc tgccccactc
gggcctgtcc ccacaggtga gtacctcctg 27420accgtgctgg catctaatgc
cttcgagaac cggacgcagc aggtgcctgt gagcgtgcgc 27480gcctccctgc
cctccgtggc tgtgggtgtg agtgacggcg tcctggtggc cggccggccc
27540gtcaccttct acccgcaccc gctgccctcg cctgggggtg ttctttacac
gtgggacttc 27600ggggacggct cccctgtcct gacccagagc cagccggctg
ccaaccacac ctatgcctcg 27660aggggcacct accacgtgcg cctggaggtc
aacaacacgg tgagcggtgc ggcggcccag 27720gcggatgtgc gcgtctttga
ggagctccgc ggactcagcg tggacatgag cctggccgtg 27780gagcagggcg
cccccgtggt ggtcagcgcc gcggtgcaga cgggcgacaa catcacgtgg
27840accttcgaca tgggggacgg caccgtgctg tcgggcccgg aggcaacagt
ggagcatgtg 27900tacctgcggg cacagaactg cacagtgacc gtgggtgcgg
ccagccccgc cggccacctg 27960gcccggagcc tgcacgtgct ggtcttcgtc
ctggaggtgc tgcgcgttga acccgccgcc 28020tgcatcccca cgcagcctga
cgcgcggctc acggcctacg tcaccgggaa cccggcccac 28080tacctcttcg
actggacctt cggggatggc tcctccaaca cgaccgtgcg ggggtgcccg
28140acggtgacac acaacttcac gcggagcggc acgttccccc tggcgctggt
gctgtccagc 28200cgcgtgaaca gggcgcatta cttcaccagc atctgcgtgg
agccagaggt gggcaacgtc 28260accctgcagc cagagaggca gtttgtgcag
ctcggggacg aggcctggct ggtggcatgt 28320gcctggcccc cgttccccta
ccgctacacc tgggactttg gcaccgagga agccgccccc 28380acccgtgcca
ggggccctga ggtgacgttc atctaccgag acccaggctc ctatcttgtg
28440acagtcaccg cgtccaacaa catctctgct gccaatgact cagccctggt
ggaggtgcag 28500gagcccgtgc tggtcaccag catcaaggtc aatggctccc
ttgggctgga gctgcagcag 28560ccgtacctgt tctctgctgt gggccgtggg
cgccccgcca gctacctgtg ggatctgggg 28620gacggtgggt ggctcgaggg
tccggaggtc acccacgctt acaacagcac aggtgacttc 28680accgttaggt
ggccggctgg aatgaggtga gccgcagcga ggcctggctc aatgtgacgg
28740tgaagcggcg cgtgcggggg ctcgtcgtca atgcaagccc cacggtggtg
cccctgaatg 28800ggagcgtgag cttcagcacg tcgctggagg ccggcagtga
tgtgcgctat tcctgggtgc 28860tctgtgaccg ctgcacgccc atccctgggg
gtcctaccat ctcttacacc ttccgctccg 28920tgggcacctt caatatcatc
gtcacggctg agaacgaggt gggctccgcc caggacagca 28980tcttcgtcta
tgtcctgcag ctcatagagg ggctgcaggt ggtgggcggt ggccgctact
29040tccccaccaa ccacacggta cagctgcagg ccgtggttag ggatggcacc
aacgtctcct 29100acagctggac tgcctggagg gacaggggcc cggccctggc
cggcagcggc aaaggcttct 29160cgctcaccgt ctcgaggccg gcacctacca
tgtgcagctg cgggccacca acatgctggg 29220cagcgcctgg gccgactgca
ccatggactt cgtggagcct gtggggtggc tgatggtggc 29280cgcctccccg
aacccagctg ccgtcaacaa aagcgtcacc ctcagtgccg agctggctgg
29340tggcagtggt gtcgtataca cttggtcctt ggaggagggg ctgagctggg
agacctccga 29400gccatttacc acccatagct tccccacacc cggcctgcac
ttggtcacca tgacggcagg 29460gaacccgctg ggctcagcca acgccaccgt
ggaagtggat gtgcaggtgc ctgtgagtgg 29520cctcagcatc agggccagcg
agcccggagg cagcttcgtg gcggccgggt cctctgtgcc 29580cttttggggg
cagctggcca cgggcaccaa tgtgagctgg tgctgggctg tgcccggcgg
29640cagcagcaag cgtggccctc atgtcaccat ggtcttcccg gatgctggca
ccttctccat 29700ccggctcaat gcctccaacg cagtcagctg ggtctcagcc
acgtacaacc tcacggcgga 29760ggagcccatc gtgggcctgg tgctgtgggc
cagcagcaag gtggtggcgc ccgggcagct 29820ggtccatttt cagatcctgc
tggctgccgg ctcagctgtc accttccgcc tgcaggtcgg 29880cggggccaac
cccgaggtgc tccccgggcc ccgtttctcc cacagcttcc cccgcgtcgg
29940agaccacgtg gtgagcgtgc ggggcaaaaa ccacgtgagc tgggcccagg
cgcaggtgcg 30000catcgtggtg ctggaggccg tgagtgggct gcaggtgccc
aactgctgcg agcctggcat 30060cgccacgggc actgagagga acttcacagc
ccgcgtgcag
cgcggctctc gggtcgccta 30120cgcctggtac ttctcgctgc agaaggtcca
gggcgactcg ctggtcatcc tgtcgggccg 30180cgacgtcacc tacacgcccg
tggccgcggg gctgttggag atccaggtgc gcgccttcaa 30240cgccctgggc
agtgagaacc gcacgctggt gctggaggtt caggacgccg tccagtatgt
30300ggccctgcag agcggcccct gcttcaccaa ccgctcggcg cagtttgagg
ccgccaccag 30360ccccagcccc cggcgtgtgg cctaccactg ggactttggg
gatgggtcgc cagggcagga 30420cacagatgag cccagggccg agcactccta
cctgaggcct ggggactacc gcgtgcaggt 30480gaacgcctcc aacctggtga
gcttcttcgt ggcgcaggcc acggtgaccg tccaggtgct 30540ggcctgccgg
gagccggagg tggacgtggt cctgcccctg caggtgctga tgcggcgatc
30600acagcgcaac tacttggagg cccacgttga cctgcgcgac tgcgtcacct
accagactga 30660gtaccgctgg gaggtgtatc gcaccgccag ctgccagcgg
ccggggcgcc cagcgcgtgt 30720ggccctgccc ggcgtggacg tgagccggcc
tcggctggtg ctgccgcggc tggcgctgcc 30780tgtggggcac tactgctttg
tgtttgtcgt gtcatttggg gacacgccac tgacacagag 30840catccaggcc
aatgtgacgg tggcccccga gcgcctggtg cccatcattg agggtggctc
30900ataccgcgtg tggtcagaca cacgggacct ggtgctggat gggagcgagt
cctacgaccc 30960caacctggag gacggcgacc agacgccgct cagtttccac
tgggcctgtg tggcttcgac 31020acaggtcagt gcgtggcagg gccgtcctcc
atgcccctca cccgtccaca cccatgagcc 31080cagagaacac ccagcttgcc
accagggctg gcccgtcctc agtgcctggt gggccccgtc 31140ccagcatggg
gagggggtct cccgcgctgt ctcctgggcc gggctctgct ttaaaactgg
31200atggggctct caggccacgt cgccccttgt tctcggcctg cagagggagg
ctggcgggtg 31260tgcgctgaac tttgggcccc gcgggagcag cacggtcacc
attccacggg agcggctggc 31320ggctggcgtg gagtacacct tcagcctgac
cgtgtggaag gccggccgca aggaggaggc 31380caccaaccag acggtgggtg
ccgcccgccc ctcggccact tgccttggac agcccagcct 31440ccctggtcat
ctactgtttt ccgtgtttta gtgctggtgg aggccgcacg ctctcccctc
31500tctgtttctg atgcaaattc tatgtaacac gacagcctgc ttcagctttg
cttccttcca 31560aacctgccac agttccacgt acagtcttca agccacatat
gctctagtgg caaaagctac 31620acagtcccct agcaatacca acagtgagga
agagcccctt cccaccccag aggtagccac 31680tgtccccagc ccatgtccct
gttgctggat gtggtgggcc ggttctcacc ctcacgctcc 31740cctctctgga
ccggccagga ggcttggtga ccctgagccc gtggtggctg ctcctgctgc
31800tgtcaggcgg ggcctgctgg tgccccagag tgggcgtctg ttccccagtc
cctgctttcc 31860tcagctggcc tgattggggg tcttcccaga ggggtcgtct
gaggggaggg tgtgggagca 31920ggttccatcc cagctcagcc tcctgaccca
ggccctggct aagggctgca ggagtctgtg 31980agtcaggcct acgtggcagc
tgcggtcctc acacccacac atacgtctct tctcacacgc 32040atccccccag
gggccctcag tgagcattgc ctgcctcctg ctagggtcca gctgggtcca
32100gtacaccaga acgcacactc cagtgtcctc tgccctgtgt atgcccttcc
gccgtccaag 32160ttggaaggtg gcaaaccgga tgagtatcct gggagggagt
gagctcaccg gcagtggcca 32220ggcccctggg aaacctggag tttgggagca
gcatcctcca tgggtccccc agtccttcca 32280gcaggccaaa tagacctgtg
ttggaggtaa ccccactccc acgccaggtg ctgatccgga 32340gtggccgggt
gcccattgtg tccttggagt gtgtgtcctg caaggcacag gccgtgtacg
32400aagtgagccg cagctcctac gtgtacttgg agggccgctg cctcaattgc
agcagcggct 32460ccaagcgagg ggtgagtgtt gagcggggtg tgggcgggct
ggggatgggt cccatggccg 32520aggggacggg gcctgcaggc agaagtgggg
ctgacagggc agagggttgc gccccctcac 32580caccccttct gcctgcagcg
gtgggctgca cgtacgttca gcaacaagac gctggtgctg 32640gatgagacca
ccacatccac gggcagtgca ggcatgcgac tggtgctgcg gcggggcgtg
32700ctgcgggacg gcgagggata caccttcacg ctcacggtgc tgggccgctc
tggcgaggag 32760gagggctgcg cctccatccg cctgtccccc aaccgcccgc
cgctgggggg ctcttgccgc 32820ctcttcccac tgggcgctgt gcacgccctc
accaccaagg tgcacttcga atgcacgggt 32880gagtgcaggc ctgcgtgggg
ggagcagcgg gatcccccga ctctgtgacg tcacggagcc 32940ctcccgtgat
gccgtgggga ccgtccctca ggctggcatg acgcggagga tgctggcgcc
33000ccgctggtgt acgccctgct gctgcggcgc tgtcgccagg gccactgcga
ggagttctgt 33060gtctacaagg gcagcctctc cagctacgga gccgtgctgc
ccccgggttt caggccacac 33120ttcgaggtgg gcctggccgt ggtggtgcag
gaccagctgg gagccgctgt ggtcgccctc 33180aacaggtgag ccaggccgtg
ggagggcgcc cccgagactg ccacctgctc accaccccct 33240ctgctcgtag
gtctttggcc atcaccctcc cagagcccaa cggcagcgca acggggctca
33300cagtctggct gcacgggctc accgctagtg tgctcccagg gctgctgcgg
caggccgatc 33360cccagcacgt catcgagtac tcgttggccc tggtcaccgt
gctgaacgag gtgagtgcag 33420cctgggaggg gacgtcacat ctgctgcatg
cgtgcttggg accaagacct gtacccctgc 33480ctggagcttt gcagagggct
catcccgggc cccagagata aatcccagtg accctgaagc 33540agcaccccga
ccttccgctc ccagcagcca cacccaccgg gccctctccg gcgtctgctt
33600tccacaatgc agcccccgcc caggagggcc catgtgctta ccctgttttg
cccatgaaga 33660aacagctcag tgttgtgggt cagtgcccgc atcacacagc
gtctagcacg taactgcacc 33720ccgggagtcg tgggcatctg ctggcctcct
gccggcctcc tgcgctgctg acagcttgct 33780gtgccccctg cctgccccag
tacgagcggg ccctggacgt ggcgcagagc ccaagcacga 33840gcggcagcac
cgagcccaga tacgcaagaa catcacggag actctggtgt ccctgagggt
33900ccacactgtg gatgacatcc agcagatcgc tgctgcgctg gcccagtgca
tggtaggatg 33960gccccacctg ctcaccctgc cccgcatgcc tgccagggca
ctgggttcag ccccccaggg 34020cagacgggca gcttggccga ggagctgagc
ctccagcctg ggctccttcc tgccatggcg 34080ttcctcggtc tctgacctgc
ttcagtagcc tcagccgttc tgtcctgtgt gaacgcaggg 34140tgcctctcgg
gggacccagg gtgtaaagag gggcccagat gtggggaggg actaagaaga
34200tgctgctctg tgccctccac tctcccctcc cctcccctcc cccttccctc
ccctagcccc 34260tcccctcctc ccctccccta gcccttcccc tcctcccctc
ccctagccct ttcccttctt 34320cccccccagc ccttcccctc ctcccctccc
ctagcccttc ccctcctccc ctcccctacc 34380ccttcccctc ctcccctccc
ctagaccttc ccctcacctc ctcccgctga gcccctccac 34440tcgtccccca
gcccctccct cccctagccc ctcccctccc ccttcctccc ctcctccccc
34500tcccctcctc cccctccctc ttcctccccc tcccctcctc ccccttcctc
ccctctcctc 34560cccctcccct cctgtccccc ctcctcccct cctccctcct
cccctcctcc cccctcctcc 34620tccccctcct ccctcctccc tcctccccct
cctcctcctc ccctcctccc tcctcccctc 34680ctcccctccc ctcctccccc
tcccccctcc cttcctcccc ctcccccctc ccctcctccc 34740cctctcctcc
tcccatccct cctcccatcc ctcctccccg ttcccattct ctcccctccc
34800ccttccattt ctccctcctc cccctgccct cctctcctcc tcacctcccc
ttctccgctc 34860ctttcttctc ctccctccct ttctctcctc cctccccttc
tccccttctc ctcttctccc 34920cttctcctct cttttcatcc ttcccttctt
ccctcctttc ctcctctttt ccctcttctc 34980ccccctcctc ccctccttcc
tcctcccatt ccccctcctc ccccctccca ttccccctcc 35040tcccctcctt
cctcctccca ttacccctcc tctcctcccc tcctcccacc cccctctcct
35100cccggctcct ctcctcccct cctcatcccc ctcctctcct tccctcctaa
cccccctcct 35160ctcctcccct cctcatcccc ctcctctcct tccctcctcc
tatcccccct cctctcctcc 35220cctcctccta ttccccctcc tctcctcccc
tccttcctcc tcctctcctc ccatgccccc 35280tcctcccctc ctcccatccc
cctcctcccc tcctccctcc tcccatccca tccccctcct 35340ctcctcccct
tctctcccct cctctcctcc cctcctctcc tctcctcctc tcctcccctc
35400ctcccatccc ccctcctccc atcccccctc ctctcctccc cactcctctc
ctccccactc 35460ctctcctccc ctcatccccc tcctctctcc tcccctcccc
ctcctctcct tccctcctcc 35520tttcctcccc tccccctcct tccccctcct
ccccctcctt ctccccatcc cccttcccct 35580tctcctcctc tcccctcccc
cttctctttt tccctcctcc tcccttcctc ctcccctctt 35640ctcccctttt
cccttttctc ttcctctcct ccccttctcc cctcctgtcc tccctccctt
35700tctctctttc tttcctccct ttccttctcc cctgttctcc tcccttccct
tctccccttt 35760tcttccctcc tcctttcctc ccctcctcct tttctctgtt
tctcttcctt tcccctccac 35820tttccccttc ctttcccctc tcctttctcc
ttcctttcct ctccccttct cttccttttc 35880ctctctcccc ttcttttccc
tcttcccctc ccctcctctt cccctcccct cctcttcccc 35940tcccctcctc
ttcccctccc ctcctcttcc cctctcctcc tcttcccctc ccctcctctt
36000tccctcccct cttctcctcc cctcctctcc cctcttcccc tcccctcctc
ttccctcccc 36060ttcccctccc ctcctcttcc ctccccttcc cctcccctcc
tcttccctcc ccttcccctc 36120ctcttccttc ctctcttccc ctcccctcct
cttccctccc ctcttcccct ccccttctct 36180tctcctcccc ttctcttccc
ctcccctttt cttccctctc cttgtcttcc ctgccctcct 36240cttccctccc
ctcctcttcc ctcccctctt cccctctcct cctcttccct cccctcttcc
36300tctttcctct tcccctcccc tcctcctccc tcccctttcc cctcttcccc
tcccctccgc 36360ttccctcccc tttctccccc ttctctcccc tcccctctcc
ccccttctct cccctcccct 36420ctcccccttc tctcccctcc cctctccccc
ttctctcccc tctcctctcc cccttctctc 36480ccccttctct cccccttctc
tctccccttc tctccccctt ctctcccctc cccccttctc 36540tcccctcccc
tctccccctt ctctcccctc ccctctcccc tgtcctctcc tctccaccct
36600tctctcccct cccctctcct ctcccccttc cctctcctct cccccttctc
tcccctcccc 36660tctcctctcc ccccttttct ccactcccct ctcctctctc
ccctcctcct ccgctctcat 36720gtgaagaggt gccttgtgtg gtcggtgggc
tgcatcacgt ggtccccagg tggaggccct 36780gggtcatgca gagccacaga
aaatgcttag tgaggaggct gtgggggtcc agtcaagtgg 36840gctctccagc
tgcagggctg ggggtgggag ccaggtgagg acccgtgtag agaggagggc
36900gtgtgcaagg agtggggcca ggagcggggc tggacactgc tggctccaca
caggggccca 36960gcagggagct cgtatgccgc tcgtgcctga agcagacgct
gcacaagctg gaggccatga 37020tgctcatcct gcaggcagag accaccgcgg
gcaccgtgac gcccaccgcc atcggagaca 37080gcatcctcaa catcacaggt
gccgcggccc gtgccccatg ccacccgccc gccccgtgcg 37140gccctttcct
ctgcctccct cctcccccca accgcgtcgc ctttgcccca tcccatcttc
37200gtccccctcc cctcccccca attcccatcc tcatccccct cccccaattc
ccattctcct 37260ccccctcccc cttccctatt accatccctt ttctccatct
ctctcccctt ttctccattt 37320ccccccccgt cctccccgtc cttttgtcca
ttcccctcat cttcctcatc cccctcatcc 37380cccttcccct cccttatccc
ccttcccctc cctttccccc tgctcctctt cttctccctt 37440ctcttttctc
tacccttttc cttccttttt cctccctctc cccatcatcc ccctcatctt
37500cgtcctcatc cccatcacct tccccctccc ccctccacca ctctctctcc
agcttccccc 37560ttccttctgc ctgcacctcg ctctctgccc cctcaggttc
cccctttctc ccagccccca 37620ccctccggct cccccttttt gcctgccccc
accctccctc tacctccctg tctctgcact 37680gacctcacgc atgtctgcag
gagacctcat ccacctggcc agctcggacg tgcgggcacc 37740acagccctca
gagctgggag ccgagtcacc atctcggatg gtggcgtccc aggcctacaa
37800cctgacctct gccctcatgc gcatcctcat gcgctcccgc gtgctcaacg
aggagcccct 37860gacgctggcg ggcgaggaga tcgtggccca gggcaagcgc
tcggacccgc ggagcctgct 37920gtgctatggc ggcgccccag ggcctggctg
ccacttctcc atccccgagg ctttcagcgg 37980ggccctggcc aacctcagtg
acgtggtgca gctcatcttt ctggtggact ccaatccctt 38040tccctttggc
tatatcagca actacaccgt ctccaccaag gtggcctcga tggcattcca
38100gacacaggcc ggcgcccaga tccccatcga gcggctggcc tcagagcgcg
ccatcaccgt 38160gaaggtgccc aacaactcgg actgggctgc ccggggccac
cgcagctccg ccaactccgc 38220caactccgtt gtggtccagc cccaggcctc
cgtcggtgct gtggtcaccc tggacagcag 38280caaccctgcg gccgggctgc
atctgcagct caactatacg ctgctggacg gtgcgtgcag 38340cgggtggggc
acacgcggcc ccctggcctt gttcttgggg ggaaggcgtt tctcgtaggg
38400cttccatggg tgtctctggt gaaatttgct ttctgtttca tgggctgctg
ggggcctggc 38460cagagaggag ctgggggcca cggagaagca ggtgccagct
ctggtgcaga ggctcctatg 38520ctttcaggcc cgtggcagag ggtgggctca
ggagggccat cgtgggtgtc ccccgggtgg 38580ttgagcttcc cggcaggcgt
gtgacctgcg cgttctgccc caggccacta cctgtctgag 38640gaacctgagc
cctacctggc agtctaccta cactcggagc cccggcccaa tgagcacaac
38700tgctcggcta gcaggaggat ccgcccagag tcactccagg gtgctgacca
ccggccctac 38760accttcttca tttccccggg gtgagctctg cgggccagcc
tggcagggca gggcagggca 38820tcatgggtca gcattgcctg ggttactggc
cccatgggga cggcaggcag cgaggggact 38880ggaccgggta tgggctctga
gactgcgaca tccaacctgg cggagcctgg gctcacgtcc 38940gctacccctt
ccctgcccag gagcagagac ccagcgggga gttaccatct gaacctctcc
39000agccacttcc gctggtcggc gctgcaggtg tccgtgggcc tgtacacgtc
cctgtgccag 39060tacttcagcg aggaggacat ggtgtggcgg acagaggggc
tgctgcccct ggaggagacc 39120tcgccccgcc aggccgtctg cctcacccgc
cacctcaccg ccttcggcgc cagcctcttc 39180gtgcccccaa gccatgtccg
ctttgtgttt cctgtgagtg accctgtgct cctgggagcc 39240tctgcagagt
cgaggagggc ctgggtgggc tcggctctat cctgagaagg cacagcttgc
39300acgtgacctc ctgggcccgg cggctgtgtc ctcacaggag ccgacagcgg
atgtaaacta 39360catcgtcatg ctgacatgtg ctgtgtgcct ggtgacctac
atggtcatgg ccgccatcct 39420gcacaagctg gaccagttgg atgccagccg
gggccgcgcc atccctttct gtgggcagcg 39480gggccgcttc aagtacgaga
tcctcgtcaa gacaggctgg ggccggggct caggtgaggg 39540gcgcagcggg
gtggcagggc ctcccctgct ctcactggct gtgctggttg caccctctgg
39600gagtgagtct cgtcgcaggc gtcagaacaa ggcagttttt gcagtgctgt
gtgaagggct 39660cgtgtgttca tcctgggaat gacctcgtga gcactcactg
tccctgagga ctaggacagc 39720tcctagctgg aagtaggtgc cagtcagtca
gggtgggcag cccacgttct gcacagtagc 39780gtggccccac aagtgacgtg
agcatcgcta ccactgtggg agactgtgca tccacccgcg 39840atcctgactg
catagctcgt ctctcagacg gaggcgccag caccctcccc gtggctgttt
39900cttcagtacc tccattttcc tttcattgga attgcccttc tggcattccc
tttttgtttt 39960cgtttttctt tttttagaga cggagtctca ctctgttgcc
caggctggag tgcaatggca 40020tgatcttggc tcacagcaac ttccagctcc
cgggtttaag ccattcccct taagcgattc 40080tcctgagtag ctgggagtac
aggtgcacac caccacaccc agttaatttt tcaccatgtc 40140agccaggcga
actcctgacc tcaggtgatc cgcctgcctc ggcctgccag agtgctggga
40200tgacaggtgt gagccaccac acctggctgt gttcccattt tttatctctg
tgctgctttc 40260ctcttcattg cccagttctt tcttttgatt acctactttt
aaaaactgtc ggccgggcgc 40320ggtggctcac acctgtaatc cgagcacttt
gggaggccag gcaggcaaat cacggggtca 40380ggagatcgag accatcctgg
ctaacggtga aaccctgtct ctaataaaaa gtacaaaaaa 40440attagcccgg
cgtagtggca ggcgcctgta gtcccagctc cttgggagac tgaggcagga
40500gaatggcgtg aacccgggag gcggagcttg cagtgagctg agattgcgcc
actgcactcc 40560agcctgggtg acacagcaag actccatctc aaaaaaaaaa
gaaaaaaaat actgtcacct 40620gggtctgtca ctgggagagg aggtgacaca
gcttcacgct ttgcagtctg tgcatgaact 40680gagggacggg tgtgtggtgc
gggtcaccgg ttgtggcatg actgaggcgt ggacaggtgt 40740gcagtgcggg
tcactggttg tggtgtggac tgaggcgtgt gcagccatgt ttgcatgtca
40800caagttacag ttctttccat gtaacttaat catgtccttg aggtcctgct
gttaattgga 40860caaattgcag taaccgcagc tccttgtgta tggcagagcc
gtgcaaagcc gggactgcct 40920gtgtggctcc ttgagtgcgc acaggccaaa
gctgagatga cttgcctggg atgccacacg 40980tgttgggcag cagaccgagc
ctcccacccc tccctcttgc ctcccaggta ccacggccca 41040cgtgggcatc
atgctgtatg gggtggacag ccggagcggc caccggcacc tggacggcga
41100cagagccttc caccgcaaca gcctggacat cttccggatc gccaccccgc
acagcctggg 41160tagcgtgtgg aagatccgag tgtggcacga caacaaaggt
ttgtgcggac cctgccaagc 41220tctgcccctc tgcccccgca ttggggcgcc
ctgcgagcct gacctccctc ctgcgcctct 41280gcagggctca gccctgcctg
gttcctgcag cacgtcatcg tcagggacct gcagacggca 41340cgcagcgcct
tcttcctggt caatgactgg ctttcggtgg agacggaggc caacgggggc
41400ctggtggaga aggaggtgct ggccgcgagt aaggcctcgt tccatggtcc
cactccgtgg 41460gaggttgggc agggtggtcc tgccccgtgg cctcctgcag
tgcggccctc cctgccttct 41520aggcgacgca gcccttttgc gcttccggcg
cctgctggtg gctgagctgc agcgtggctt 41580ctttgacaag cacatctggc
tctccatatg ggaccggccg cctcgtagcc gtttcactcg 41640catccagagg
gccacctgct gcgttctcct catctgcctc ttcctgggcg ccaacgccgt
41700gtggtacggg gctgttggcg actctgccta caggtgggtg ccgtaggggt
cggggcagcc 41760tcttcctgcc cagcccttcc tgcccctcag cctcacctgt
gtggcctcct ctcctccaca 41820cagcacgggg catgtgtcca ggctgagccc
gctgagcgtc gacacagtcg ctgttggcct 41880ggtgtccagc gtggttgtct
atcccgtcta cctggccatc ctttttctct tccggatgtc 41940ccggagcaag
gtgggctggg gctggggacc cgggagtact gggaatggag cctgggcctc
42000ggcaccatgc ctagggccgc cactttccag tgctgcagcc agagggaaag
gcgtccacca 42060aaggctgctc gggaagggtc aacacacttg agcagcctta
gctagactga ccagggagaa 42120agagagaaga ctcagaagcc agaatggtga
aagaacgagg gcactttgct aagcagacgc 42180cacggacgac tgcacagcag
cacgccagat aactcagaag aagcaagcac gcggctgtgc 42240acgcttccga
aatgcactcc agaagaaaat ctcagtacat ctataggaag tgaagaggct
42300gagttagtcc cttagaaacg tcccagtggc cgggccgggt gtggtggctc
acgcctgtaa 42360tcccaacact tcaggtggcc gaggtgggcg gatctgagtc
caggagtttg agaccagcct 42420gggcaacata gcaagacccc atctatataa
aacattaaaa agggccaggc gcggtggctc 42480acgcctgtaa tcccagcact
ttgggaggcc gaggcgggca gatcacttga ggtcaggagt 42540tcgagaccag
cctggccaac acaatgaaac cccgactcta ctacaaatac aaaaacttag
42600ctgggcatgg tggcgggcgc ctgtagtccc agctactcga gaggctgagg
caggagaatg 42660gcatgaaccc aggaggcgga gcttgcagtg agccgagatt
gcgccactgc actccatcct 42720gggcaacgga gcaagactcc atctccaaaa
aaaaaaaaaa aaaatcccac aaagaaaagc 42780tcaggctcag agccttcacg
atagaatttt tctaagcagt taaggaagaa ttaacaccaa 42840tccttcacag
actctttcca agaatacagc aggtgggaac gcttcccatt catacggaaa
42900cgggaggccg caccccttag gaatgcacac gtggggtcct caagaggtta
catgcaaact 42960aaccccagca gcacacagag aaggcgcata agccgcgacc
aggaggggtt gctcccgagt 43020ccgtggcagg aaccagaggc cacatgtggc
tgctcgtatt taagttaatt aaaatggaac 43080gatggccggg tgtggtggct
cacacctgta atcccagcac tttgggaggc ggaggcgggc 43140agatcacttg
aggtcaggag ttccaagacc agcctggcca acacagtgaa accccgtctc
43200tactaaaaat acaaaaaatt agctgggcat ggtggcaggc acctgtaatc
ccagctactc 43260aggaggctga gccaggacaa tcgcctgaac gcgggaggtg
gaggttgcag tgagctgaga 43320ttgcgccatt gcactccagc ctgggtgaca
gcgagactcc atctaaaaaa gaaaatatga 43380aatttaaaac tctgttcctt
agctgcacca gtctgctgtc aagtgttcag tggcacacgt 43440cgcgaggggc
tgccatcacg gacggtgcag atgtcccata tatccagcat tctaggacat
43500tctgtcagat ggcaccgggc tctgtcctgt ctgctgagga ggtggcttct
catccctgtc 43560ctgagcaggt ctgagctgcc gcccgctgac cactgccctc
gtcctgcagg tggctgggag 43620cccgagcccc acacctgccg ggcagcaggt
gctggacatc gacagctgcc tggactcgtc 43680cgtgctggac agctccttcc
tcacgttctc aggcctccac gctgaggtga ggactctact 43740gggggtcctg
ggctgggctg ggggtcctgc cgccttggcg cagcttggac tcaagacact
43800gtgcacctct cagcaggcct ttgttggaca gatgaagagt gacttgtttc
tggatgattc 43860taagaggtgg gttccctaga gaaacctcga gccctggtgc
aggtcactgt gtctggggtg 43920ccgggggtgt gcgggctgcg tgtccttgct
gggtgtctgt ggctccatgt ggtcacacca 43980cccgggagca ggtttgctcg
gaagcccagg gtgtccgtgc gtgactggac gggggtgggc 44040tgtgtgtgtg
acacatcccc tggtaccttg ctgacccgcg ccacctgcag tctggtgtgc
44100tggccctccg gcgagggaac gctcagttgg ccggacctgc tcagtgaccc
gtccattgtg 44160ggtagcaatc tgcggcagct ggcacggggc caggcgggcc
atgggctggg cccagaggag 44220gacggcttct ccctggccag cccctactcg
cctgccaaat ccttctcagc atcaggtgag 44280ctggggtgag aggagggggc
tctgaagctc acccttgcag ctgggcccac cctatgcctc 44340ctgtacctct
agatgaagac ctgatccagc aggtccttgc cgagggggtc agcagcccag
44400cccctaccca agacacccac atggaaacgg acctgctcag cagcctgtga
gtgtccggct 44460ctcgggggag gggggattgc cagaggaggg gccgggactc
aggccaggca gccgtggttc 44520ccgcctgggg tagggtgggg tggggtgcca
gggcagggct gtggctgcac cacttcactt 44580ctctgaacct ctgttgtctg
tggaaagagc ctcatgggat ccccagggcc ccagaacctt 44640ccctctaggg
agggagcagg ctcatggggc tttgtaggag cagaaaggct cctgtgtgag
44700gctggccggg gccacgtttt tatcttggtc tcagagcagt gagaaattat
gggcgggttt 44760ttaaataccc catttttggc cgggcgcggt ggctcacacg
tgtaatccca gcactttggg 44820aggccgaggt gggcagatga cctgaggtca
gcagttcgag accagcctgg ccaacatggc 44880gaaaccccgt ctctactaaa
aatacaaaaa attagccggg catgctggca ggcgcctgta 44940gtcccagtta
ctcgggagac tgaggtagga gaatcgattg aacctggtag gtgaaggttg
45000tagtgagccg agatcgcgcc actgcactcc agcctgggca acaagagcga
aactccgtct 45060caaaaacaaa aaaattcctc aatttcttgg ttgttttgta
acttatcaac aaatggtcat 45120atagaggtta ccttgtatgt agtcacgcac
atagtcacgc
acatggcagc cggcggcgga 45180gcgcacccac ggcgtgttcc cacgcgtgtg
accccgggct ctgccatgcc ctcctatgct 45240caggtgtgct gaggtccaca
cggccctgcc gttgcactgc agctgcctgc aggattcagt 45300gcagtggcat
gcagtgcagg tgcggtgccc cggagccaca ggccacacca cagggcctgc
45360atgcacaggg gctgcggtgt ctgggtttgg gtaactacgc cctgtgacat
ttgcacagca 45420acagaattac ctaatgacgc atttctcaga acacatccct
ggcactaagt ggtgcgtgac 45480tgctgctttt gcatccacat ctagtttgat
ttgtgtgtta ttcctttgag tgcttctcat 45540tgttaagcaa ccaagaacta
aagaggtatg aactgcccct ggactcaaac aaaaaggaaa 45600acttcctgat
ttacaaaagg cagataacca tcacatgagg gcatctttat gaataaattg
45660ctggttggtt ttaaaaatac agagtatggg gaaatccagg ggtagtcact
acatgctgac 45720cagccccagg tatctccggc ccaaagctct gtgaaatcca
gattcagtgc ttccgcgggg 45780atttctgacg gcagctcaga ctccgcatcc
acacagagcg cgtggccctc accctcccgg 45840cttcctcaac ccttggccgt
cccttgctcg gacagtgctt cgggctgacc aggtcggagg 45900cttgggtttg
tcctggaccc ctctgcgtcc ttcctcactg cagcctccag cgcgtcccgt
45960ggctcctttc ccaacgcaga gcacggcctt ccctgcgcct gagcctgcac
cctccgtcct 46020ggcggcgcct ctgccctggc attccctgcc actccatgcc
tccctattgg ccattctccg 46080tctctgccag cgagagcctg ctccctgagt
cagaccctga gtcatttgtg ttgctataaa 46140ggaatagttg aggctgggtt
attttttatt tttatttatt tttttgagat ggagtctctg 46200ttgcccagac
tggagtgcag tcgcatgatc tcggctcact gcaaagtctg cctcccacgt
46260tcaagcagtt atctgcctca gcctcccaag tagctaagat tacaggcgcc
cgccgccaca 46320gccggctaat tttttgtgtg tgtgttttag tagagaggag
gtttcaccat cttagccagg 46380ctggtcttga actcctgacc tcgtgatcca
cccatctcag cctcccaaaa tgctgagatt 46440acaggcgtga gccaccacgc
ctgaccaagt tgaggctagg tcatttttta attttttgta 46500aagacagggt
ctcactgtct ccaactcctg agctcaagtg atcctcctgc ctcagcctcc
46560tgaagtgctg ggattacagg cttgagacac tgcgcccagc caagagtgtc
ttttatcctc 46620cgagagacag caaaacagga agcattcagt gcagtgtgac
cctgggtcag gccgttcttt 46680cggtgatggg ctgacgaggg cgcaggtacg
ggagagcgtc ctgagagccc gggactcggc 46740gtctcgcagt tggtctcgtc
ctccccctca acgtgtcttc gctgcctctg tacctcttct 46800ctagcagctc
tgggaccggg catatcagca tggtggcccg atgcagtggc acagcctcgg
46860tggtcactgg ctcctggaga cacaagcaga tctctggcct cagggagccc
tacacactgt 46920tgggatttga aaggcattca tatgtttcct tgtccagaag
ttaattttag gccataaacc 46980tgcatgggac agacacactg gcgtctctag
attgtagaga tgcttgttgg atggttgaga 47040cccaatcata gtttgcaggg
ttgaaggggg gctcattgca ccctgagaga ctgtgcactg 47100ctgtaagggc
agctggtcag gctgtgggcg atgggtttat cagcagcaag cgggcgggag
47160agggacgcag gcggacgcct gacttcggtg cctggagtgg ctcttggttc
cctggctccc 47220agcaccactc ccactctcgt ttggggtagg gtcttccggc
tttttgtcgg ggggaccctg 47280tgacccaaga ggctcaagaa actgcccgcc
caggttaaca tgggcttggc tgcaactgcc 47340tcctggaggc cgggatgaat
tcacagccta ccatgtccct caggtccagc actcctgggg 47400agaagacaga
gacgctggcg ctgcagaggc tgggggagct ggggccaccc agcccaggcc
47460tgaactggga acagccccag gcagcgaggc tgtccaggac aggtgtgctt
gcgtagcccc 47520gggatgcccc tagcccctcc ctgtgagctg cctctcacag
gtctgtctct gcttccccag 47580gactggtgga gggtctgcgg aagcgcctgc
tgccggcctg gtgtgcctcc ctggcccacg 47640ggctcagcct gctcctggtg
gctgtggctg tggctgtctc agggtgggtg ggtgcgagct 47700tccccccggg
cgtgagtgtt gcgtggctcc tgtccagcag cgccagcttc ctggcctcat
47760tcctcggctg ggagccactg aaggtgaggg ggctgccagg ggtaggctac
aggcctccat 47820cacgggggac ccctctgaag ccaccccctc cccaggtctt
gctggaagcc ctgtacttct 47880cactggtggc caagcggctg cacccggatg
aagatgacac cctggtagag agcccggctg 47940tgacgcctgt gagcgcacgt
gtgccccgcg tacggccacc ccacggcttt gcactcttcc 48000tggccaagga
agaagcccgc aaggtcaaga ggctacatgg catgctgcgg gtgagcctgg
48060gtgcggcctg tgcccctgcc acctccgtct cttgtctccc acctcccacc
catgcacgca 48120ggacactcct gtcccccttt cctcacctca gaaggccctt
aggggttcaa tgctctgcag 48180cctttgcccg gtctccctcc taccccacgc
cccccacttg ctgccccagt ccctgccagg 48240gcccagctcc aatgcccact
cctgcctggc cctgaaggcc cctaagcacc actgcagtgg 48300cctgtgtgtc
tgcccccagg tggggttccg ggcagggtgt gtgctgccat taccctggcc
48360aggtagagtc ttggggcgcc ccctgccagc tcaccttcct gcagccacac
ctgccgcagc 48420catggctcca gccgttgcca aagccctgct gtcactgtgg
gctggggcca ggctgaccac 48480agggcccccc cgtccaccag agcctcctgg
tgtacatgct ttttctgctg gtgaccctgc 48540tggccagcta tggggatgcc
tcatgccatg ggcacgccta ccgtctgcaa agcgccatca 48600agcaggagct
gcacagccgg gccttcctgg ccatcacgcg gtacgggcat ccggtgcact
48660ggtctgtctt ctgggcttta gttttgcctt tagtccagcc agaccctagg
ggacatgtgg 48720acatgtgtag atacctttgt ggctgctaga actggaggta
ggtgctgctg gcatcagtag 48780gcagagggga gggacacagg tccgtgtctt
gcagtgcaca ggacgggccc atgacagaca 48840actgtctgcc ccagaacatc
cccaggataa ggctgagaag cccaggtcta gccgtggcca 48900gcagggcagt
gggagccatg ttccctgggt ctctggtggc cgctcactcg aggcgggcat
48960ggggcagtag gggctggagc gtgtgactga tgctgtggca ggtctgagga
gctctggcca 49020tggatggccc acgtgctgct gccctacgtc cacgggaacc
agtccagccc agagctgggg 49080cccccacggc tgcggcaggt gcggctgcag
gaaggtgagc tggcagggcg tgccccaaga 49140cttaaatcgt tcctcttgtt
gagagagcag cctttagcgg agctctggca tcagccctgc 49200tccctagctg
tgtgaccttt gccctcttaa caccgccgtt tccttctctg tatatgagag
49260atggtaacgt tgtctaattg atggctgctg ggagggttcc ctggggtggc
gccgaaccag 49320agctcaggcg agctggccag caggaaacac tcctgttggg
ttttgatgag gccctggccc 49380cggcctgggg ctctgtgtgt ttcagcactc
tacccagacc ctcccggccc cagggtccac 49440acgtgctcgg ccgcaggagg
cttcagcacc agcgattacg acgttggctg ggagagtcct 49500cacaatggct
cggggacgtg ggcctattca gcgccggatc tgctggggtg agcagagcga
49560gggccccggg cgtctacgcc aaggacaagg gagtagttct ccaggagtgc
cgcggcctcc 49620tgaccagcct ggctccgggg tgccggaagg gctggggtgc
ggcacccacg ccacccctct 49680ccggcagggc atggtcctgg ggctcctgtg
ccgtgtatga cagcgggggc tacgtgcagg 49740agctgggcct gagcctggag
gagagccgcg accggctgcg cttcctgcag ctgcacaact 49800ggctggacaa
caggtgggag ctccctcccc tgccctctcc ggggtggccg cagtcaccag
49860ccaggagccc accctcactc ctccggcccc cgctggccta ggcggcttcc
acagcccctc 49920agccacgcct gcactgcgcg gtccccgcag ctcccgccct
gccacccgct cctactgacc 49980cgcaccctct gcgcaggagc cgcgctgtgt
tcctggagct cacgcgctac agcccggccg 50040tggggctgca cgccgccgtc
acgctgcgcc tcgagttccc ggcggccggc cgcgccctgg 50100ccgccctcag
cgtccgcccc tttgcgctgc gccgcctcag cgcgggcctc tcgctgcctc
50160tgctcacctc ggtacgcccg tccccggcca gaccccgcgc ctcccaccgg
cagcgtcccg 50220ccccctcgcg gggccccgcc cggcagcgtc tcacccctcg
cagcgccccg ccccctcgca 50280gcgtcccgcc ccctcgcagg gccccgcccc
ggcagcgtcc cgccccctcg tagggccccg 50340ccccggcagc gtcccgcccc
ctcgcagggc cccgccccgg cagcgtccct cccgccctcc 50400tgaccgcgcc
ccccacaggt gtgcctgctg ctgttcgccg tgcacttcgc cgtggccgag
50460gcccgtactt ggcacaggga agggcgctgg cgcgtgctgc ggctcggagc
ctgggcgcgg 50520tggctgctgg tggcgctgac ggcggccacg gcactggtac
gcctcgccca gctgggtgcc 50580gctgaccgcc agtggacccg tttcgtgcgc
ggccgcccgc gccgcttcac tagcttcgac 50640caggtggcgc agctgagctc
cgcagcccgt ggcctggcgg cctcgctgct cttcctgctt 50700ttggtcaagg
tgagggctgg gccggtgggc gcggggctgg gcgcacaccc cagggctgca
50760agcagacaga tttctcgtcc gcaggctgcc cagcagctac gcttcgtgcg
ccagtggtcc 50820gtctttggca agacattatg ccgagctctg ccagagctcc
tgggggtcac cttgggcctg 50880gtggtgctcg gggtagccta cgcccagctg
gccatcctgg taggtgactg cgcggccggg 50940gagggcgtct tagctcagct
cagctcagct gtacgccctc actggtgtcg ccttccccgc 51000agctcgtgtc
ttcctgtgtg gactccctct ggagcgtggc ccaggccctg ttggtgctgt
51060gccctgggac tgggctctct accctgtgtc ctgccgagtc ctggcacctg
tcacccctgc 51120tgtgtgtggg gctctgggca ctgcggctgt ggggcgccct
acggctgggg gctgttattc 51180tccgctggcg ctaccacgcc ttgcgtggag
agctgtaccg gccggcctgg gagccccagg 51240actacgagat ggtggagttg
ttcctgcgca ggctgcgcct ctggatgggc ctcagcaagg 51300tcaaggaggt
gggtacggcc cagtgggggg gagagggaca cgccctgggc tctgcccagg
51360gtgcagccgg actgactgag cccctgtgcc gcccccagtt ccgccacaaa
gtccgctttg 51420aagggatgga gccgctgccc tctcgctcct ccaggggctc
caaggtatcc ccggatgtgc 51480ccccacccag cgctggctcc gatgcctcgc
acccctccac ctcctccagc cagctggatg 51540ggctgagcgt gagcctgggc
cggctgggga caaggtgtga gcctgagccc tcccgcctcc 51600aagccgtgtt
cgaggccctg ctcacccagt ttgaccgact caaccaggcc acagaggacg
51660tctaccagct ggagcagcag ctgcacagcc tgcaaggccg caggagcagc
cgggcgcccg 51720ccggatcttc ccgtggccca tccccgggcc tgcggccagc
actgcccagc cgccttgccc 51780gggccagtcg gggtgtggac ctggccactg
gccccagcag gacacccctt cgggccaaga 51840acaaggtcca ccccagcagc
acttagtcct ccttcctggc gggggtgggc cgtggagtcg 51900gagtggacac
cgctcagtat tactttctgc cgctgtcaag gccgagggcc aggcagaatg
51960gctgcacgta ggttccccag agagcaggca ggggcatctg tctgtctgtg
ggcttcagca 52020ctttaaagag gctgtgtggc caaccaggac ccagggtccc
ctccccagct cccttgggaa 52080ggacacagca gtattggacg gtttctagcc
tctgagatgc taatttattt ccccgagtcc 52140tcaggtacag cgggctgtgc
ccggccccac cccctgggca gatgtccccc actgctaagg 52200ctgctggctt
cagggagggt tagcctgcac cgccgccacc ctgcccctaa gttattacct
52260ctccagttcc taccgtactc cctgcaccgt ctcactgtgt gtctcgtgtc
agtaatttat 52320atggtgttaa aatgtgtata tttttgtatg tcactatttt
cactagggct gaggggcctg 52380cgcccagagc tggcctcccc caacacctgc
tgcgcttggt aggtgtggtg gcgttatggc 52440agcccggctg ctgcttggat
gcgagcttgg ccttgggccg gtgctggggg cacagctgtc 52500tgccaggcac
tctcatcacc ccagaggcct tgtcatcctc ccttgcccca ggccaggtag
52560caagagagca gcgcccaggc ctgctggcat caggtctggg caagtagcag
gactaggcat 52620gtcagaggac cccagggtgg ttagaggaaa agactcctcc
tgggggctgg ctcccagggt 52680ggaggaaggt gactgtgtgt gtgtgtgtgt
gcgcgcgcgc acgcgcgagt gtgctgtatg 52740gcccaggcag cctcaaggcc
ctcggagctg gctgtgcctg cttctgtgta ccacttctgt 52800gggcatggcc
gcttctagag cctcgacacc cccccaaccc ccgcaccaag cagacaaagt
52860caataaaaga gctgtctgac tgcaatctgt gcctctatgt ctgtgcactg
gggtcaggac 52920tttatttatt tcactgacag gcaataccgt ccaaggccag
tgcaggaggg agggccccgg 52980cctcacacaa actcggtgaa gtcctccacc
gaggagatga ggcgcttccg ctggcccacc 53040tcatagccag gtgtgggctc
ggctggagtc tgtgcagggg ctttgctatg ggacggaggg 53100tgcaccagag
gtaggctggg gttggagtag gcggcttcct cgcagatctg aaggcagagg
53160cggcttgggc agtaagtctg ggaggcgtgg caaccgctct gcccacacac
ccgccccaca 53220gcttgggcag ccagcacacc ccgctgaggg agccccatat
tccctacccg ctggcggagc 53280gcttgatgtg gcggagcggg caatccactt
ggaggggtag atatcggtgg ggttggagcg 53340gctatgatgc acctgtgagg
ccatctgggg acgtaggcag ggggtgagct cactatcagg 53400tggcacctgg
gcctgtccca ccagctcacg cctggaccca cccccactca catttgcgtg
53460cagggccatc tggcgggcca cgaagggcag gttgcggtca gacacgatct
tggccacgct 53520gg 5352224303PRTHomo sapiens 2Met Pro Pro Ala Ala
Pro Ala Arg Leu Ala Leu Ala Leu Gly Leu Gly 1 5 10 15 Leu Trp Leu
Gly Ala Leu Ala Gly Gly Pro Gly Arg Gly Cys Gly Pro 20 25 30 Cys
Glu Pro Pro Cys Leu Cys Gly Pro Ala Pro Gly Ala Ala Cys Arg 35 40
45 Val Asn Cys Ser Gly Arg Gly Leu Arg Thr Leu Gly Pro Ala Leu Arg
50 55 60 Ile Pro Ala Asp Ala Thr Glu Leu Asp Val Ser His Asn Leu
Leu Arg 65 70 75 80 Ala Leu Asp Val Gly Leu Leu Ala Asn Leu Ser Ala
Leu Ala Glu Leu 85 90 95 Asp Ile Ser Asn Asn Lys Ile Ser Thr Leu
Glu Glu Gly Ile Phe Ala 100 105 110 Asn Leu Phe Asn Leu Ser Glu Ile
Asn Leu Ser Gly Asn Pro Phe Glu 115 120 125 Cys Asp Cys Gly Leu Ala
Trp Leu Pro Gln Trp Ala Glu Glu Gln Gln 130 135 140 Val Arg Val Val
Gln Pro Glu Ala Ala Thr Cys Ala Gly Pro Gly Ser 145 150 155 160 Leu
Ala Gly Gln Pro Leu Leu Gly Ile Pro Leu Leu Asp Ser Gly Cys 165 170
175 Gly Glu Glu Tyr Val Ala Cys Leu Pro Asp Asn Ser Ser Gly Thr Val
180 185 190 Ala Ala Val Ser Phe Ser Ala Ala His Glu Gly Leu Leu Gln
Pro Glu 195 200 205 Ala Cys Ser Ala Phe Cys Phe Ser Thr Gly Gln Gly
Leu Ala Ala Leu 210 215 220 Ser Glu Gln Gly Trp Cys Leu Cys Gly Ala
Ala Gln Pro Ser Ser Ala 225 230 235 240 Ser Phe Ala Cys Leu Ser Leu
Cys Ser Gly Pro Pro Ala Pro Pro Ala 245 250 255 Pro Thr Cys Arg Gly
Pro Thr Leu Leu Gln His Val Phe Pro Ala Ser 260 265 270 Pro Gly Ala
Thr Leu Val Gly Pro His Gly Pro Leu Ala Ser Gly Gln 275 280 285 Leu
Ala Ala Phe His Ile Ala Ala Pro Leu Pro Val Thr Asp Thr Arg 290 295
300 Trp Asp Phe Gly Asp Gly Ser Ala Glu Val Asp Ala Ala Gly Pro Ala
305 310 315 320 Ala Ser His Arg Tyr Val Leu Pro Gly Arg Tyr His Val
Thr Ala Val 325 330 335 Leu Ala Leu Gly Ala Gly Ser Ala Leu Leu Gly
Thr Asp Val Gln Val 340 345 350 Glu Ala Ala Pro Ala Ala Leu Glu Leu
Val Cys Pro Ser Ser Val Gln 355 360 365 Ser Asp Glu Ser Leu Asp Leu
Ser Ile Gln Asn Arg Gly Gly Ser Gly 370 375 380 Leu Glu Ala Ala Tyr
Ser Ile Val Ala Leu Gly Glu Glu Pro Ala Arg 385 390 395 400 Ala Val
His Pro Leu Cys Pro Ser Asp Thr Glu Ile Phe Pro Gly Asn 405 410 415
Gly His Cys Tyr Arg Leu Val Val Glu Lys Ala Ala Trp Leu Gln Ala 420
425 430 Gln Glu Gln Cys Gln Ala Trp Ala Gly Ala Ala Leu Ala Met Val
Asp 435 440 445 Ser Pro Ala Val Gln Arg Phe Leu Val Ser Arg Val Thr
Arg Ser Leu 450 455 460 Asp Val Trp Ile Gly Phe Ser Thr Val Gln Gly
Val Glu Val Gly Pro 465 470 475 480 Ala Pro Gln Gly Glu Ala Phe Ser
Leu Glu Ser Cys Gln Asn Trp Leu 485 490 495 Pro Gly Glu Pro His Pro
Ala Thr Ala Glu His Cys Val Arg Leu Gly 500 505 510 Pro Thr Gly Trp
Cys Asn Thr Asp Leu Cys Ser Ala Pro His Ser Tyr 515 520 525 Val Cys
Glu Leu Gln Pro Gly Gly Pro Val Gln Asp Ala Glu Asn Leu 530 535 540
Leu Val Gly Ala Pro Ser Gly Asp Leu Gln Gly Pro Leu Thr Pro Leu 545
550 555 560 Ala Gln Gln Asp Gly Leu Ser Ala Pro His Glu Pro Val Glu
Val Met 565 570 575 Val Phe Pro Gly Leu Arg Leu Ser Arg Glu Ala Phe
Leu Thr Thr Ala 580 585 590 Glu Phe Gly Thr Gln Glu Leu Arg Arg Pro
Ala Gln Leu Arg Leu Gln 595 600 605 Val Tyr Arg Leu Leu Ser Thr Ala
Gly Thr Pro Glu Asn Gly Ser Glu 610 615 620 Pro Glu Ser Arg Ser Pro
Asp Asn Arg Thr Gln Leu Ala Pro Ala Cys 625 630 635 640 Met Pro Gly
Gly Arg Trp Cys Pro Gly Ala Asn Ile Cys Leu Pro Leu 645 650 655 Asp
Ala Ser Cys His Pro Gln Ala Cys Ala Asn Gly Cys Thr Ser Gly 660 665
670 Pro Gly Leu Pro Gly Ala Pro Tyr Ala Leu Trp Arg Glu Phe Leu Phe
675 680 685 Ser Val Pro Ala Gly Pro Pro Ala Gln Tyr Ser Val Thr Leu
His Gly 690 695 700 Gln Asp Val Leu Met Leu Pro Gly Asp Leu Val Gly
Leu Gln His Asp 705 710 715 720 Ala Gly Pro Gly Ala Leu Leu His Cys
Ser Pro Ala Pro Gly His Pro 725 730 735 Gly Pro Arg Ala Pro Tyr Leu
Ser Ala Asn Ala Ser Ser Trp Leu Pro 740 745 750 His Leu Pro Ala Gln
Leu Glu Gly Thr Trp Gly Cys Pro Ala Cys Ala 755 760 765 Leu Arg Leu
Leu Ala Gln Arg Glu Gln Leu Thr Val Leu Leu Gly Leu 770 775 780 Arg
Pro Asn Pro Gly Leu Arg Leu Pro Gly Arg Tyr Glu Val Arg Ala 785 790
795 800 Glu Val Gly Asn Gly Val Ser Arg His Asn Leu Ser Cys Ser Phe
Asp 805 810 815 Val Val Ser Pro Val Ala Gly Leu Arg Val Ile Tyr Pro
Ala Pro Arg 820 825 830 Asp Gly Arg Leu Tyr Val Pro Thr Asn Gly Ser
Ala Leu Val Leu Gln 835 840 845 Val Asp Ser Gly Ala Asn Ala Thr Ala
Thr Ala Arg Trp Pro Gly Gly 850 855 860 Ser Leu Ser Ala Arg Phe Glu
Asn Val Cys Pro Ala Leu Val Ala Thr 865 870 875 880 Phe Val Pro Ala
Cys Pro Trp Glu Thr Asn Asp Thr Leu Phe Ser Val 885 890 895 Val Ala
Leu Pro Trp Leu Ser Glu Gly Glu His Val Val Asp Val Val 900 905 910
Val Glu Asn Ser Ala Ser Arg Ala Asn Leu Ser Leu Arg Val Thr Ala 915
920 925 Glu Glu Pro Ile Cys Gly Leu Arg Ala Thr Pro Ser Pro Glu Ala
Arg 930 935 940 Val Leu Gln Gly Val Leu Val Arg Tyr Ser Pro Val Val
Glu Ala Gly 945 950 955 960 Ser Asp Met Val Phe Arg Trp Thr Ile Asn
Asp Lys Gln Ser Leu Thr 965 970 975 Phe Gln Asn Val Val Phe Asn Val
Ile Tyr Gln Ser Ala Ala Val Phe 980 985 990 Lys Leu Ser Leu Thr Ala
Ser Asn His Val Ser Asn Val Thr Val Asn 995 1000 1005 Tyr Asn Val
Thr Val Glu Arg Met Asn Arg Met
Gln Gly Leu Gln 1010 1015 1020 Val Ser Thr Val Pro Ala Val Leu Ser
Pro Asn Ala Thr Leu Ala 1025 1030 1035 Leu Thr Ala Gly Val Leu Val
Asp Ser Ala Val Glu Val Ala Phe 1040 1045 1050 Leu Trp Thr Phe Gly
Asp Gly Glu Gln Ala Leu His Gln Phe Gln 1055 1060 1065 Pro Pro Tyr
Asn Glu Ser Phe Pro Val Pro Asp Pro Ser Val Ala 1070 1075 1080 Gln
Val Leu Val Glu His Asn Val Thr His Thr Tyr Ala Ala Pro 1085 1090
1095 Gly Glu Tyr Leu Leu Thr Val Leu Ala Ser Asn Ala Phe Glu Asn
1100 1105 1110 Leu Thr Gln Gln Val Pro Val Ser Val Arg Ala Ser Leu
Pro Ser 1115 1120 1125 Val Ala Val Gly Val Ser Asp Gly Val Leu Val
Ala Gly Arg Pro 1130 1135 1140 Val Thr Phe Tyr Pro His Pro Leu Pro
Ser Pro Gly Gly Val Leu 1145 1150 1155 Tyr Thr Trp Asp Phe Gly Asp
Gly Ser Pro Val Leu Thr Gln Ser 1160 1165 1170 Gln Pro Ala Ala Asn
His Thr Tyr Ala Ser Arg Gly Thr Tyr His 1175 1180 1185 Val Arg Leu
Glu Val Asn Asn Thr Val Ser Gly Ala Ala Ala Gln 1190 1195 1200 Ala
Asp Val Arg Val Phe Glu Glu Leu Arg Gly Leu Ser Val Asp 1205 1210
1215 Met Ser Leu Ala Val Glu Gln Gly Ala Pro Val Val Val Ser Ala
1220 1225 1230 Ala Val Gln Thr Gly Asp Asn Ile Thr Trp Thr Phe Asp
Met Gly 1235 1240 1245 Asp Gly Thr Val Leu Ser Gly Pro Glu Ala Thr
Val Glu His Val 1250 1255 1260 Tyr Leu Arg Ala Gln Asn Cys Thr Val
Thr Val Gly Ala Gly Ser 1265 1270 1275 Pro Ala Gly His Leu Ala Arg
Ser Leu His Val Leu Val Phe Val 1280 1285 1290 Leu Glu Val Leu Arg
Val Glu Pro Ala Ala Cys Ile Pro Thr Gln 1295 1300 1305 Pro Asp Ala
Arg Leu Thr Ala Tyr Val Thr Gly Asn Pro Ala His 1310 1315 1320 Tyr
Leu Phe Asp Trp Thr Phe Gly Asp Gly Ser Ser Asn Thr Thr 1325 1330
1335 Val Arg Gly Cys Pro Thr Val Thr His Asn Phe Thr Arg Ser Gly
1340 1345 1350 Thr Phe Pro Leu Ala Leu Val Leu Ser Ser Arg Val Asn
Arg Ala 1355 1360 1365 His Tyr Phe Thr Ser Ile Cys Val Glu Pro Glu
Val Gly Asn Val 1370 1375 1380 Thr Leu Gln Pro Glu Arg Gln Phe Val
Gln Leu Gly Asp Glu Ala 1385 1390 1395 Trp Leu Val Ala Cys Ala Trp
Pro Pro Phe Pro Tyr Arg Tyr Thr 1400 1405 1410 Trp Asp Phe Gly Thr
Glu Glu Ala Ala Pro Thr Arg Ala Arg Gly 1415 1420 1425 Pro Glu Val
Thr Phe Ile Tyr Arg Asp Pro Gly Ser Tyr Leu Val 1430 1435 1440 Thr
Val Thr Ala Ser Asn Asn Ile Ser Ala Ala Asn Asp Ser Ala 1445 1450
1455 Leu Val Glu Val Gln Glu Pro Val Leu Val Thr Ser Ile Lys Val
1460 1465 1470 Asn Gly Ser Leu Gly Leu Glu Leu Gln Gln Pro Tyr Leu
Phe Ser 1475 1480 1485 Ala Val Gly Arg Gly Arg Pro Ala Ser Tyr Leu
Trp Asp Leu Gly 1490 1495 1500 Asp Gly Gly Trp Leu Glu Gly Pro Glu
Val Thr His Ala Tyr Asn 1505 1510 1515 Ser Thr Gly Asp Phe Thr Val
Arg Val Ala Gly Trp Asn Glu Val 1520 1525 1530 Ser Arg Ser Glu Ala
Trp Leu Asn Val Thr Val Lys Arg Arg Val 1535 1540 1545 Arg Gly Leu
Val Val Asn Ala Ser Arg Thr Val Val Pro Leu Asn 1550 1555 1560 Gly
Ser Val Ser Phe Ser Thr Ser Leu Glu Ala Gly Ser Asp Val 1565 1570
1575 Arg Tyr Ser Trp Val Leu Cys Asp Arg Cys Thr Pro Ile Pro Gly
1580 1585 1590 Gly Pro Thr Ile Ser Tyr Thr Phe Arg Ser Val Gly Thr
Phe Asn 1595 1600 1605 Ile Ile Val Thr Ala Glu Asn Glu Val Gly Ser
Ala Gln Asp Ser 1610 1615 1620 Ile Phe Val Tyr Val Leu Gln Leu Ile
Glu Gly Leu Gln Val Val 1625 1630 1635 Gly Gly Gly Arg Tyr Phe Pro
Thr Asn His Thr Val Gln Leu Gln 1640 1645 1650 Ala Val Val Arg Asp
Gly Thr Asn Val Ser Tyr Ser Trp Thr Ala 1655 1660 1665 Trp Arg Asp
Arg Gly Pro Ala Leu Ala Gly Ser Gly Lys Gly Phe 1670 1675 1680 Ser
Leu Thr Val Leu Glu Ala Gly Thr Tyr His Val Gln Leu Arg 1685 1690
1695 Ala Thr Asn Met Leu Gly Ser Ala Trp Ala Asp Cys Thr Met Asp
1700 1705 1710 Phe Val Glu Pro Val Gly Trp Leu Met Val Ala Ala Ser
Pro Asn 1715 1720 1725 Pro Ala Ala Val Asn Thr Ser Val Thr Leu Ser
Ala Glu Leu Ala 1730 1735 1740 Gly Gly Ser Gly Val Val Tyr Thr Trp
Ser Leu Glu Glu Gly Leu 1745 1750 1755 Ser Trp Glu Thr Ser Glu Pro
Phe Thr Thr His Ser Phe Pro Thr 1760 1765 1770 Pro Gly Leu His Leu
Val Thr Met Thr Ala Gly Asn Pro Leu Gly 1775 1780 1785 Ser Ala Asn
Ala Thr Val Glu Val Asp Val Gln Val Pro Val Ser 1790 1795 1800 Gly
Leu Ser Ile Arg Ala Ser Glu Pro Gly Gly Ser Phe Val Ala 1805 1810
1815 Ala Gly Ser Ser Val Pro Phe Trp Gly Gln Leu Ala Thr Gly Thr
1820 1825 1830 Asn Val Ser Trp Cys Trp Ala Val Pro Gly Gly Ser Ser
Lys Arg 1835 1840 1845 Gly Pro His Val Thr Met Val Phe Pro Asp Ala
Gly Thr Phe Ser 1850 1855 1860 Ile Arg Leu Asn Ala Ser Asn Ala Val
Ser Trp Val Ser Ala Thr 1865 1870 1875 Tyr Asn Leu Thr Ala Glu Glu
Pro Ile Val Gly Leu Val Leu Trp 1880 1885 1890 Ala Ser Ser Lys Val
Val Ala Pro Gly Gln Leu Val His Phe Gln 1895 1900 1905 Ile Leu Leu
Ala Ala Gly Ser Ala Val Thr Phe Arg Leu Gln Val 1910 1915 1920 Gly
Gly Ala Asn Pro Glu Val Leu Pro Gly Pro Arg Phe Ser His 1925 1930
1935 Ser Phe Pro Arg Val Gly Asp His Val Val Ser Val Arg Gly Lys
1940 1945 1950 Asn His Val Ser Trp Ala Gln Ala Gln Val Arg Ile Val
Val Leu 1955 1960 1965 Glu Ala Val Ser Gly Leu Gln Val Pro Asn Cys
Cys Glu Pro Gly 1970 1975 1980 Ile Ala Thr Gly Thr Glu Arg Asn Phe
Thr Ala Arg Val Gln Arg 1985 1990 1995 Gly Ser Arg Val Ala Tyr Ala
Trp Tyr Phe Ser Leu Gln Lys Val 2000 2005 2010 Gln Gly Asp Ser Leu
Val Ile Leu Ser Gly Arg Asp Val Thr Tyr 2015 2020 2025 Thr Pro Val
Ala Ala Gly Leu Leu Glu Ile Gln Val Arg Ala Phe 2030 2035 2040 Asn
Ala Leu Gly Ser Glu Asn Arg Thr Leu Val Leu Glu Val Gln 2045 2050
2055 Asp Ala Val Gln Tyr Val Ala Leu Gln Ser Gly Pro Cys Phe Thr
2060 2065 2070 Asn Arg Ser Ala Gln Phe Glu Ala Ala Thr Ser Pro Ser
Pro Arg 2075 2080 2085 Arg Val Ala Tyr His Trp Asp Phe Gly Asp Gly
Ser Pro Gly Gln 2090 2095 2100 Asp Thr Asp Glu Pro Arg Ala Glu His
Ser Tyr Leu Arg Pro Gly 2105 2110 2115 Asp Tyr Arg Val Gln Val Asn
Ala Ser Asn Leu Val Ser Phe Phe 2120 2125 2130 Val Ala Gln Ala Thr
Val Thr Val Gln Val Leu Ala Cys Arg Glu 2135 2140 2145 Pro Glu Val
Asp Val Val Leu Pro Leu Gln Val Leu Met Arg Arg 2150 2155 2160 Ser
Gln Arg Asn Tyr Leu Glu Ala His Val Asp Leu Arg Asp Cys 2165 2170
2175 Val Thr Tyr Gln Thr Glu Tyr Arg Trp Glu Val Tyr Arg Thr Ala
2180 2185 2190 Ser Cys Gln Arg Pro Gly Arg Pro Ala Arg Val Ala Leu
Pro Gly 2195 2200 2205 Val Asp Val Ser Arg Pro Arg Leu Val Leu Pro
Arg Leu Ala Leu 2210 2215 2220 Pro Val Gly His Tyr Cys Phe Val Phe
Val Val Ser Phe Gly Asp 2225 2230 2235 Thr Pro Leu Thr Gln Ser Ile
Gln Ala Asn Val Thr Val Ala Pro 2240 2245 2250 Glu Arg Leu Val Pro
Ile Ile Glu Gly Gly Ser Tyr Arg Val Trp 2255 2260 2265 Ser Asp Thr
Arg Asp Leu Val Leu Asp Gly Ser Glu Ser Tyr Asp 2270 2275 2280 Pro
Asn Leu Glu Asp Gly Asp Gln Thr Pro Leu Ser Phe His Trp 2285 2290
2295 Ala Cys Val Ala Ser Thr Gln Arg Glu Ala Gly Gly Cys Ala Leu
2300 2305 2310 Asn Phe Gly Pro Arg Gly Ser Ser Thr Val Thr Ile Pro
Arg Glu 2315 2320 2325 Arg Leu Ala Ala Gly Val Glu Tyr Thr Phe Ser
Leu Thr Val Trp 2330 2335 2340 Lys Ala Gly Arg Lys Glu Glu Ala Thr
Asn Gln Thr Val Leu Ile 2345 2350 2355 Arg Ser Gly Arg Val Pro Ile
Val Ser Leu Glu Cys Val Ser Cys 2360 2365 2370 Lys Ala Gln Ala Val
Tyr Glu Val Ser Arg Ser Ser Tyr Val Tyr 2375 2380 2385 Leu Glu Gly
Arg Cys Leu Asn Cys Ser Ser Gly Ser Lys Arg Gly 2390 2395 2400 Arg
Trp Ala Ala Arg Thr Phe Ser Asn Lys Thr Leu Val Leu Asp 2405 2410
2415 Glu Thr Thr Thr Ser Thr Gly Ser Ala Gly Met Arg Leu Val Leu
2420 2425 2430 Arg Arg Gly Val Leu Arg Asp Gly Glu Gly Tyr Thr Phe
Thr Leu 2435 2440 2445 Thr Val Leu Gly Arg Ser Gly Glu Glu Glu Gly
Cys Ala Ser Ile 2450 2455 2460 Arg Leu Ser Pro Asn Arg Pro Pro Leu
Gly Gly Ser Cys Arg Leu 2465 2470 2475 Phe Pro Leu Gly Ala Val His
Ala Leu Thr Thr Lys Val His Phe 2480 2485 2490 Glu Cys Thr Gly Trp
His Asp Ala Glu Asp Ala Gly Ala Pro Leu 2495 2500 2505 Val Tyr Ala
Leu Leu Leu Arg Arg Cys Arg Gln Gly His Cys Glu 2510 2515 2520 Glu
Phe Cys Val Tyr Lys Gly Ser Leu Ser Ser Tyr Gly Ala Val 2525 2530
2535 Leu Pro Pro Gly Phe Arg Pro His Phe Glu Val Gly Leu Ala Val
2540 2545 2550 Val Val Gln Asp Gln Leu Gly Ala Ala Val Val Ala Leu
Asn Arg 2555 2560 2565 Ser Leu Ala Ile Thr Leu Pro Glu Pro Asn Gly
Ser Ala Thr Gly 2570 2575 2580 Leu Thr Val Trp Leu His Gly Leu Thr
Ala Ser Val Leu Pro Gly 2585 2590 2595 Leu Leu Arg Gln Ala Asp Pro
Gln His Val Ile Glu Tyr Ser Leu 2600 2605 2610 Ala Leu Val Thr Val
Leu Asn Glu Tyr Glu Arg Ala Leu Asp Val 2615 2620 2625 Ala Ala Glu
Pro Lys His Glu Arg Gln His Arg Ala Gln Ile Arg 2630 2635 2640 Lys
Asn Ile Thr Glu Thr Leu Val Ser Leu Arg Val His Thr Val 2645 2650
2655 Asp Asp Ile Gln Gln Ile Ala Ala Ala Leu Ala Gln Cys Met Gly
2660 2665 2670 Pro Ser Arg Glu Leu Val Cys Arg Ser Cys Leu Lys Gln
Thr Leu 2675 2680 2685 His Lys Leu Glu Ala Met Met Leu Ile Leu Gln
Ala Glu Thr Thr 2690 2695 2700 Ala Gly Thr Val Thr Pro Thr Ala Ile
Gly Asp Ser Ile Leu Asn 2705 2710 2715 Ile Thr Gly Asp Leu Ile His
Leu Ala Ser Ser Asp Val Arg Ala 2720 2725 2730 Pro Gln Pro Ser Glu
Leu Gly Ala Glu Ser Pro Ser Arg Met Val 2735 2740 2745 Ala Ser Gln
Ala Tyr Asn Leu Thr Ser Ala Leu Met Arg Ile Leu 2750 2755 2760 Met
Arg Ser Arg Val Leu Asn Glu Glu Pro Leu Thr Leu Ala Gly 2765 2770
2775 Glu Glu Ile Val Ala Gln Gly Lys Arg Ser Asp Pro Arg Ser Leu
2780 2785 2790 Leu Cys Tyr Gly Gly Ala Pro Gly Pro Gly Cys His Phe
Ser Ile 2795 2800 2805 Pro Glu Ala Phe Ser Gly Ala Leu Ala Asn Leu
Ser Asp Val Val 2810 2815 2820 Gln Leu Ile Phe Leu Val Asp Ser Asn
Pro Phe Pro Phe Gly Tyr 2825 2830 2835 Ile Ser Asn Tyr Thr Val Ser
Thr Lys Val Ala Ser Met Ala Phe 2840 2845 2850 Gln Thr Gln Ala Gly
Ala Gln Ile Pro Ile Glu Arg Leu Ala Ser 2855 2860 2865 Glu Arg Ala
Ile Thr Val Lys Val Pro Asn Asn Ser Asp Trp Ala 2870 2875 2880 Ala
Arg Gly His Arg Ser Ser Ala Asn Ser Ala Asn Ser Val Val 2885 2890
2895 Val Gln Pro Gln Ala Ser Val Gly Ala Val Val Thr Leu Asp Ser
2900 2905 2910 Ser Asn Pro Ala Ala Gly Leu His Leu Gln Leu Asn Tyr
Thr Leu 2915 2920 2925 Leu Asp Gly His Tyr Leu Ser Glu Glu Pro Glu
Pro Tyr Leu Ala 2930 2935 2940 Val Tyr Leu His Ser Glu Pro Arg Pro
Asn Glu His Asn Cys Ser 2945 2950 2955 Ala Ser Arg Arg Ile Arg Pro
Glu Ser Leu Gln Gly Ala Asp His 2960 2965 2970 Arg Pro Tyr Thr Phe
Phe Ile Ser Pro Gly Ser Arg Asp Pro Ala 2975 2980 2985 Gly Ser Tyr
His Leu Asn Leu Ser Ser His Phe Arg Trp Ser Ala 2990 2995 3000 Leu
Gln Val Ser Val Gly Leu Tyr Thr Ser Leu Cys Gln Tyr Phe 3005 3010
3015 Ser Glu Glu Asp Met Val Trp Arg Thr Glu Gly Leu Leu Pro Leu
3020 3025 3030 Glu Glu Thr Ser Pro Arg Gln Ala Val Cys Leu Thr Arg
His Leu 3035 3040 3045 Thr Ala Phe Gly Ala Ser Leu Phe Val Pro Pro
Ser His Val Arg 3050 3055 3060 Phe Val Phe Pro Glu Pro Thr Ala Asp
Val Asn Tyr Ile Val Met 3065 3070 3075 Leu Thr Cys Ala Val Cys Leu
Val Thr Tyr Met Val Met Ala Ala 3080 3085 3090 Ile Leu His Lys Leu
Asp Gln Leu Asp Ala Ser Arg Gly Arg Ala 3095 3100 3105 Ile Pro Phe
Cys Gly Gln Arg Gly Arg Phe Lys Tyr Glu Ile Leu 3110 3115 3120 Val
Lys Thr Gly Trp Gly Arg Gly Ser Gly Thr Thr Ala His Val 3125 3130
3135 Gly Ile Met Leu Tyr Gly Val Asp Ser Arg Ser Gly His Arg His
3140 3145 3150 Leu Asp Gly Asp Arg Ala Phe His Arg Asn Ser Leu Asp
Ile Phe 3155 3160 3165 Arg Ile Ala Thr Pro His Ser Leu Gly Ser Val
Trp Lys Ile Arg 3170 3175 3180 Val Trp His Asp Asn Lys Gly Leu Ser
Pro Ala Trp Phe Leu Gln 3185 3190 3195 His Val Ile Val Arg Asp Leu
Gln Thr Ala Arg Ser Ala Phe Phe 3200 3205
3210 Leu Val Asn Asp Trp Leu Ser Val Glu Thr Glu Ala Asn Gly Gly
3215 3220 3225 Leu Val Glu Lys Glu Val Leu Ala Ala Ser Asp Ala Ala
Leu Leu 3230 3235 3240 Arg Phe Arg Arg Leu Leu Val Ala Glu Leu Gln
Arg Gly Phe Phe 3245 3250 3255 Asp Lys His Ile Trp Leu Ser Ile Trp
Asp Arg Pro Pro Arg Ser 3260 3265 3270 Arg Phe Thr Arg Ile Gln Arg
Ala Thr Cys Cys Val Leu Leu Ile 3275 3280 3285 Cys Leu Phe Leu Gly
Ala Asn Ala Val Trp Tyr Gly Ala Val Gly 3290 3295 3300 Asp Ser Ala
Tyr Ser Thr Gly His Val Ser Arg Leu Ser Pro Leu 3305 3310 3315 Ser
Val Asp Thr Val Ala Val Gly Leu Val Ser Ser Val Val Val 3320 3325
3330 Tyr Pro Val Tyr Leu Ala Ile Leu Phe Leu Phe Arg Met Ser Arg
3335 3340 3345 Ser Lys Val Ala Gly Ser Pro Ser Pro Thr Pro Ala Gly
Gln Gln 3350 3355 3360 Val Leu Asp Ile Asp Ser Cys Leu Asp Ser Ser
Val Leu Asp Ser 3365 3370 3375 Ser Phe Leu Thr Phe Ser Gly Leu His
Ala Glu Gln Ala Phe Val 3380 3385 3390 Gly Gln Met Lys Ser Asp Leu
Phe Leu Asp Asp Ser Lys Ser Leu 3395 3400 3405 Val Cys Trp Pro Ser
Gly Glu Gly Thr Leu Ser Trp Pro Asp Leu 3410 3415 3420 Leu Ser Asp
Pro Ser Ile Val Gly Ser Asn Leu Arg Gln Leu Ala 3425 3430 3435 Arg
Gly Gln Ala Gly His Gly Leu Gly Pro Glu Glu Asp Gly Phe 3440 3445
3450 Ser Leu Ala Ser Pro Tyr Ser Pro Ala Lys Ser Phe Ser Ala Ser
3455 3460 3465 Asp Glu Asp Leu Ile Gln Gln Val Leu Ala Glu Gly Val
Ser Ser 3470 3475 3480 Pro Ala Pro Thr Gln Asp Thr His Met Glu Thr
Asp Leu Leu Ser 3485 3490 3495 Ser Leu Ser Ser Thr Pro Gly Glu Lys
Thr Glu Thr Leu Ala Leu 3500 3505 3510 Gln Arg Leu Gly Glu Leu Gly
Pro Pro Ser Pro Gly Leu Asn Trp 3515 3520 3525 Glu Gln Pro Gln Ala
Ala Arg Leu Ser Arg Thr Gly Leu Val Glu 3530 3535 3540 Gly Leu Arg
Lys Arg Leu Leu Pro Ala Trp Cys Ala Ser Leu Ala 3545 3550 3555 His
Gly Leu Ser Leu Leu Leu Val Ala Val Ala Val Ala Val Ser 3560 3565
3570 Gly Trp Val Gly Ala Ser Phe Pro Pro Gly Val Ser Val Ala Trp
3575 3580 3585 Leu Leu Ser Ser Ser Ala Ser Phe Leu Ala Ser Phe Leu
Gly Trp 3590 3595 3600 Glu Pro Leu Lys Val Leu Leu Glu Ala Leu Tyr
Phe Ser Leu Val 3605 3610 3615 Ala Lys Arg Leu His Pro Asp Glu Asp
Asp Thr Leu Val Glu Ser 3620 3625 3630 Pro Ala Val Thr Pro Val Ser
Ala Arg Val Pro Arg Val Arg Pro 3635 3640 3645 Pro His Gly Phe Ala
Leu Phe Leu Ala Lys Glu Glu Ala Arg Lys 3650 3655 3660 Val Lys Arg
Leu His Gly Met Leu Arg Ser Leu Leu Val Tyr Met 3665 3670 3675 Leu
Phe Leu Leu Val Thr Leu Leu Ala Ser Tyr Gly Asp Ala Ser 3680 3685
3690 Cys His Gly His Ala Tyr Arg Leu Gln Ser Ala Ile Lys Gln Glu
3695 3700 3705 Leu His Ser Arg Ala Phe Leu Ala Ile Thr Arg Ser Glu
Glu Leu 3710 3715 3720 Trp Pro Trp Met Ala His Val Leu Leu Pro Tyr
Val His Gly Asn 3725 3730 3735 Gln Ser Ser Pro Glu Leu Gly Pro Pro
Arg Leu Arg Gln Val Arg 3740 3745 3750 Leu Gln Glu Ala Leu Tyr Pro
Asp Pro Pro Gly Pro Arg Val His 3755 3760 3765 Thr Cys Ser Ala Ala
Gly Gly Phe Ser Thr Ser Asp Tyr Asp Val 3770 3775 3780 Gly Trp Glu
Ser Pro His Asn Gly Ser Gly Thr Trp Ala Tyr Ser 3785 3790 3795 Ala
Pro Asp Leu Leu Gly Ala Trp Ser Trp Gly Ser Cys Ala Val 3800 3805
3810 Tyr Asp Ser Gly Gly Tyr Val Gln Glu Leu Gly Leu Ser Leu Glu
3815 3820 3825 Glu Ser Arg Asp Arg Leu Arg Phe Leu Gln Leu His Asn
Trp Leu 3830 3835 3840 Asp Asn Arg Ser Arg Ala Val Phe Leu Glu Leu
Thr Arg Tyr Ser 3845 3850 3855 Pro Ala Val Gly Leu His Ala Ala Val
Thr Leu Arg Leu Glu Phe 3860 3865 3870 Pro Ala Ala Gly Arg Ala Leu
Ala Ala Leu Ser Val Arg Pro Phe 3875 3880 3885 Ala Leu Arg Arg Leu
Ser Ala Gly Leu Ser Leu Pro Leu Leu Thr 3890 3895 3900 Ser Val Cys
Leu Leu Leu Phe Ala Val His Phe Ala Val Ala Glu 3905 3910 3915 Ala
Arg Thr Trp His Arg Glu Gly Arg Trp Arg Val Leu Arg Leu 3920 3925
3930 Gly Ala Trp Ala Arg Trp Leu Leu Val Ala Leu Thr Ala Ala Thr
3935 3940 3945 Ala Leu Val Arg Leu Ala Gln Leu Gly Ala Ala Asp Arg
Gln Trp 3950 3955 3960 Thr Arg Phe Val Arg Gly Arg Pro Arg Arg Phe
Thr Ser Phe Asp 3965 3970 3975 Gln Val Ala His Val Ser Ser Ala Ala
Arg Gly Leu Ala Ala Ser 3980 3985 3990 Leu Leu Phe Leu Leu Leu Val
Lys Ala Ala Gln His Val Arg Phe 3995 4000 4005 Val Arg Gln Trp Ser
Val Phe Gly Lys Thr Leu Cys Arg Ala Leu 4010 4015 4020 Pro Glu Leu
Leu Gly Val Thr Leu Gly Leu Val Val Leu Gly Val 4025 4030 4035 Ala
Tyr Ala Gln Leu Ala Ile Leu Leu Val Ser Ser Cys Val Asp 4040 4045
4050 Ser Leu Trp Ser Val Ala Gln Ala Leu Leu Val Leu Cys Pro Gly
4055 4060 4065 Thr Gly Leu Ser Thr Leu Cys Pro Ala Glu Ser Trp His
Leu Ser 4070 4075 4080 Pro Leu Leu Cys Val Gly Leu Trp Ala Leu Arg
Leu Trp Gly Ala 4085 4090 4095 Leu Arg Leu Gly Ala Val Ile Leu Arg
Trp Arg Tyr His Ala Leu 4100 4105 4110 Arg Gly Glu Leu Tyr Arg Pro
Ala Trp Glu Pro Gln Asp Tyr Glu 4115 4120 4125 Met Val Glu Leu Phe
Leu Arg Arg Leu Arg Leu Trp Met Gly Leu 4130 4135 4140 Ser Lys Val
Lys Glu Phe Arg His Lys Val Arg Phe Glu Gly Met 4145 4150 4155 Glu
Pro Leu Pro Ser Arg Ser Ser Arg Gly Ser Lys Val Ser Pro 4160 4165
4170 Asp Val Pro Pro Pro Ser Ala Gly Ser Asp Ala Ser His Pro Ser
4175 4180 4185 Thr Ser Ser Ser Gln Leu Asp Gly Leu Ser Val Ser Leu
Gly Arg 4190 4195 4200 Leu Gly Thr Arg Cys Glu Pro Glu Pro Ser Arg
Leu Gln Ala Val 4205 4210 4215 Phe Glu Ala Leu Leu Thr Gln Phe Asp
Arg Leu Asn Gln Ala Thr 4220 4225 4230 Glu Asp Val Tyr Gln Leu Glu
Gln Gln Leu His Ser Leu Gln Gly 4235 4240 4245 Arg Arg Ser Ser Arg
Ala Pro Ala Gly Ser Ser Arg Gly Pro Ser 4250 4255 4260 Pro Gly Leu
Arg Pro Ala Leu Pro Ser Arg Leu Ala Arg Ala Ser 4265 4270 4275 Arg
Gly Val Asp Leu Ala Thr Gly Pro Ser Arg Thr Pro Leu Arg 4280 4285
4290 Ala Lys Asn Lys Val His Pro Ser Ser Thr 4295 4300
329DNAArtificial sequencePCR primer BPF14 3ccatccacct gctgtgtgac
ctggtaaat 29426DNAArtificial sequencePCR primer BPR9 4ccacctcatc
gccccttcct aagcat 26531DNAArtificial sequencePCR primer BPF9
5attttttgag atggagcttc actcttgcag g 31620DNAArtificial sequencePCR
primer BPR4 6cgctcggcag gcccctaacc 20721DNAArtificial sequencePCR
primer BPF12 7ccgcccccag gagcctagac g 21827DNAArtificial
sequencePCR primer BPR5 8catcctgttc atccgctcca cggttac
27920DNAArtificial sequencePCR primer F13 9tggagggagg gacgccaatc
201020DNAArtificial sequencePCR primer R27 10gtcaacgtgg gcctccaagt
201121DNAArtificial sequencePCR primer F26 11agcgcaacta cttggaggcc
c 211228DNAArtificial sequencePCR primer R2 12gcagggtgag caggtggggc
catcctac 281326DNAArtificial sequencePCR primer BPF15 13gaggctgtgg
gggtccagtc aagtgg 261425DNAArtificial sequencePCR primer BPR12
14agggaggcag aggaaagggc cgaac 251524DNAArtificial sequencePCR
primer BPF6 15ccccgtcctc cccgtccttt tgtc 241621DNAArtificial
sequencePCR primer BPR6 16aagcgcaaaa gggctgcgtc g
211722DNAArtificial sequencePCR primer BPF13 17ggccctccct
gccttctagg cg 221821DNAArtificial sequencePCR primer KG8R25
18gttgcagcca agcccatgtt a 211919DNAArtificial sequencePCR primer
1F1 19ggtcgcgctg tggcgaagg 192016DNAArtificial sequencePCR primer
1R1 20cggcgggcgg catcgt 162116DNAArtificial sequencePCR primer 1F2
21acggcggggc catgcg 162218DNAArtificial sequencePCR primer 1R2
22gcgtcctggc ccgcgtcc 182320DNAArtificial sequencePCR primer 2F
23ttggggatgc tggcaatgtg 202420DNAArtificial sequencePCR primer 2R
24gggattcggc aaagctgatg 202520DNAArtificial sequencePCR primer 3F
25ccatcagctt tgccgaatcc 202620DNAArtificial sequencePCR primer 3R
26agggcagaag ggatattggg 202720DNAArtificial sequencePCR primer 4F
27agacccttcc caccagacct 202819DNAArtificial sequencePCR primer 4R
28tgagccctgc ccagtgtct 192921DNAArtificial sequencePCR primer 5F1
29gagccaggag gagcagaacc c 213021DNAArtificial sequencePCR primer
5R1 30agagggacag gcaggcaaag g 213118DNAArtificial sequencePCR
primer 5F2 31cccagccctc cagtgcct 183220DNAArtificial sequencePCR
primer 5R2 32cccaggcagc acatagcgat 203318DNAArtificial sequencePCR
primer 5F3 33ccgaggtgga tgccgctg 183421DNAArtificial sequencePCR
primer 5R3 34gaaggggagt gggcagcaga c 213521DNAArtificial
sequencePCR primer 6F 35cactgaccgt tgacaccctc g 213621DNAArtificial
sequencePCR primer 6R 36tgccccagtg cttcagagat c 213719DNAArtificial
sequencePCR primer 7F 37ggagtgccct gagccccct 193819DNAArtificial
sequencePCR primer 7R 38cccctaacca cagccagcg 193921DNAArtificial
sequencePCR primer 8F 39tctgttcgtc ctggtgtcct g 214021DNAArtificial
sequencePCR primer 8R 40gcaggagggc aggttgtaga a 214120DNAArtificial
sequencePCR primer 9F 41ggtaggggga gtctgggctt 204217DNAArtificial
sequencePCR primer 9R 42gaggccaccc cgagtcc 174320DNAArtificial
sequencePCR primer 10F 43gttgggcatc tctgacggtg 204420DNAArtificial
sequencePCR primer 10R 44ggaaggtggc ctgaggagat 204517DNAArtificial
sequencePCR primer 11F2 45ggggtccacg ggccatg 174620DNAArtificial
sequencePCR primer 11R2 46aagcccagca gcacggtgag 204717DNAArtificial
sequencePCR primer 11midF 47gcttgcagcc acggaac 174820DNAArtificial
sequencePCR primer 11midR 48gcagtgctac cactgagaac
204923DNAArtificial sequencePCR primer 11F1 49tgcccctggg agaccaacga
tac 235022DNAArtificial sequencePCR primer 11R1 50ggctgctgcc
ctcactggga ag 225118DNAArtificial sequencePCR primer 12F
51gaggcgacag gctaaggg 185225DNAArtificial sequencePrimer for PCR
52aggtcaacgt gggcctccaa gtagt 255319DNAArtificial sequenceForward
nested primer F32 53gccttgcgca gcttggact 195420DNAArtificial
sequenceSecond specific primer 31R 54acagtgtctt gagtccaagc
205530DNAArtificial sequencePCR primer 55ctggtgacct acatggtcat
ggccgagatc 305630DNAArtificial sequencePCR primer 56ggttgtctat
cccgtctacc tggccctcct 305725DNAArtificial sequencePCR primer
57gtccccagcc ccagcccacc tggcc 25587PRTHomo sapiens 58Trp Asp Phe
Gly Asp Gly Ser 1 5 594PRTHomo sapiens 59His Leu Thr Ala 1
6027DNAArtificial sequencePCR primer 60gcagggtgag caggtggggc
catccta 276119DNAArtificial sequencePCR primer 12R-2 61catgaagcag
agcagaagg 196220DNAArtificial sequencePCR primer 13F 62tggagggagg
gacgccaatc 206319DNAArtificial sequencePCR primer 13R 63gaggctgggg
ctgggacaa 196418DNAArtificial sequencePCR primer 14F 64cccggttcac
tcactgcg 186520DNAArtificial sequencePCR primer 14R 65ccgtgctcag
agcctgaaag 206618DNAArtificial sequencePCR primer 15F16
66cgggtgggga gcaggtgg 186721DNAArtificial sequencePCR primer 15R16
67gctctgggtc aggacagggg a 216818DNAArtificial sequencePCR primer
15F15 68cgcctggggg tgttcttt 186918DNAArtificial sequencePCR primer
15R15 69acgtgatgtt gtcgcccg 187018DNAArtificial sequencePCR primer
15F14 70gcccccgtgg tggtcagc 187118DNAArtificial sequencePCR primer
15R14 71caggctgcgt ggggatgc 187218DNAArtificial sequencePCR primer
15F13 72ctggaggtgc tgcgcgtt 187318DNAArtificial sequencePCR primer
15R13 73ctggctccac gcagatgc 187418DNAArtificial sequencePCR primer
15F12 74cgtgaacagg gcgcatta 187521DNAArtificial sequencePCR primer
15R12 75gcagcagaga tgttgttgga c 217621DNAArtificial sequencePCR
primer 15F11 76ccaggctcct atcttgtgac a 217721DNAArtificial
sequencePCR primer 15R11 77tgaagtcacc tgtgctgttg t
217819DNAArtificial sequencePCR primer 15F10 78ctacctgtgg gatctgggg
197918DNAArtificial sequencePCR primer 15R10 79tgctgaagct cacgctcc
188020DNAArtificial
sequencePCR primer 15F9 80gggctcgtcg tcaatgcaag 208120DNAArtificial
sequencePCR primer 15R9 81caccacctgc agcccctcta 208220DNAArtificial
sequencePCR primer 15F8 82ccgcccagga cagcatcttc 208318DNAArtificial
sequencePCR primer 15R8 83cgctgcccag catgttgg 188419DNAArtificial
sequencePCR primer 15F7 84cggcaaaggc ttctcgctc 198520DNAArtificial
sequencePCR primer 15R7 85ccgggtgtgg ggaagctatg 208621DNAArtificial
sequencePCR primer 15F6 86cgagccattt accacccata g
218719DNAArtificial sequencePCR primer 15R6 87gcccagcacc agctcacat
198819DNAArtificial sequencePCR primer 15F5 88ccacgggcac caatgtgag
198920DNAArtificial sequencePCR primer 15R5 89ggcagccagc aggatctgaa
209018DNAArtificial sequencePCR pimer 15F4 90cagcagcaag gtggtggc
189118DNAArtificial sequencePCR primer 15R4 91gcgtaggcga cccgagag
189221DNAArtificial sequencePCR primer 15F3 92acgggcactg agaggaactt
c 219320DNAArtificial sequencePCR primer 15R3 93accagcgtgc
ggttctcact 209419DNAArtificial sequencePCR primer 15F2 94gccgcgacgt
cacctacac 199518DNAArtificial sequencePCR primer 15R2 95tcggccctgg
gctcatct 189620DNAArtificial sequencePCR primer 15F1 96gtcgccaggg
caggacacag 209721DNAArtificial sequencePCR primer 15F1-1
97acttggaggc ccacgttgac c 219819DNAArtificial sequencePCR primer
15R1-1 98tgatgggcac caggcgctc 199921DNAArtificial sequencePCR
primer 15F1-2 99catccaggcc aatgtgacgg t 2110021DNAArtificial
sequencePCR primer 15R1-2 100cctggtggca agctgggtgt t
2110120DNAArtificial sequencePCR primer 16F 101taaaactgga
tggggctctc 2010218DNAArtificial sequencePCR primer 16R
102ggcctccacc agcactaa 1810320DNAArtificial sequencePCR primer 17F
103gggtccccca gtccttccag 2010417DNAArtificial sequencePCR primer
17R 104tccccagccc gcccaca 1710520DNAArtificial sequencePCR primer
18F 105gccccctcac caccccttct 2010618DNAArtificial sequencePCR
primer 18R 106tcccgctgct ccccccac 1810718DNAArtificial sequencePCR
primer 19F 107gatgccgtgg ggaccgtc 1810820DNAArtificial sequencePCR
primer 19R 108gtgagcaggt ggcagtctcg 2010921DNAArtificial
sequencePCR primer 20F 109ccaccccctc tgctcgtagg t
2111019DNAArtificial sequencePCR primer 20R 110ggtcccaagc acgcatgca
1911122DNAArtificial sequencePCR primer 21F 111tgccggcctc
ctgcgctgct ga 2211228DNAArtificial sequencePCR primer TWR2-1
112gtaggatggc cccacctgct caccctgc 2811320DNAArtificial sequencePCR
primer R27 113aggtcaacgt gggcctccaa 20
* * * * *