U.S. patent application number 10/627253 was filed with the patent office on 2004-08-19 for polymorphisms in the human gene for the multidrug resistance-associated protein 1 (mrp-1) and their use in diagnostic and therapeutic applications.
Invention is credited to Brinkmann, Ulrich, Hoffmeyer, Sven, Mornhinweg, Ester.
Application Number | 20040161768 10/627253 |
Document ID | / |
Family ID | 8176294 |
Filed Date | 2004-08-19 |
United States Patent
Application |
20040161768 |
Kind Code |
A1 |
Brinkmann, Ulrich ; et
al. |
August 19, 2004 |
Polymorphisms in the human gene for the multidrug
resistance-associated protein 1 (MRP-1) and their use in diagnostic
and therapeutic applications
Abstract
The present invention relates to a polymorphic MRP-1
polynucleotide. Moreover, the invention relates to genes or vectors
comprising the polynucleotides of the invention and to a host cell
genetically engineered with the polynucleotide or gene of the
invention. Further, the invention relates to methods for producing
molecular variant polypeptides or fragments thereof, methods for
producing cells capable of expressing a molecular variant
polypeptide and to a polypeptide or fragment thereof encoded by the
polynucleotide or the gene of the invention or which is obtainable
by the method or from the cells produced by the method of the
invention. Furthermore, the invention relates to an antibody which
binds specifically the polypeptide of the invention. Moreover, the
invention relates to a transgenic non-human animal. The invention
also relates to a solid support comprising one or a plurality of
the above mentioned polynucleotides, genes, vectors, polypeptides,
antibodies or host cells. Furthermore, methods of identifying a
polymorphism, identifying and obtaining a pro-drug or drug or an
inhibitor are also encompassed by the present invention. In
addition, the invention relates to methods for producing of a
pharmaceutical composition and to methods of diagnosing a disease.
Further, the invention relates to a method of detection of the
polynucleotide of the invention. Furthermore, comprised by the
present invention are a diagnostic and a pharmaceutical
composition. Even more, the invention relates to uses of the
polynucleotides, genes, vectors, polypeptides or antibodies of the
invention. Finally, the invention relates to a diagnostic kit.
Inventors: |
Brinkmann, Ulrich;
(Weilheim, DE) ; Hoffmeyer, Sven; (Eberfing,
DE) ; Mornhinweg, Ester; (Weilheim, DE) |
Correspondence
Address: |
FISH & NEAVE
1251 AVENUE OF THE AMERICAS
50TH FLOOR
NEW YORK
NY
10020-1105
US
|
Family ID: |
8176294 |
Appl. No.: |
10/627253 |
Filed: |
July 24, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10627253 |
Jul 24, 2003 |
|
|
|
PCT/EP02/00794 |
Jan 25, 2002 |
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Current U.S.
Class: |
435/6.12 ;
435/196; 435/320.1; 435/325; 435/69.1; 536/23.2 |
Current CPC
Class: |
G01N 2800/44 20130101;
C07K 14/47 20130101; G01N 2333/705 20130101; C12Q 2600/106
20130101; C12Q 1/6883 20130101; A61P 35/00 20180101; G01N 33/6893
20130101; C12Q 2600/156 20130101; G01N 2500/04 20130101; C12Q
2600/158 20130101 |
Class at
Publication: |
435/006 ;
435/069.1; 435/196; 435/320.1; 435/325; 536/023.2 |
International
Class: |
C12Q 001/68; C07H
021/04; C12N 009/16 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 26, 2001 |
EP |
01101651.6 |
Claims
1. A polynucleotide comprising a polynucleotide selected from the
group consisting of: (a) a polynucleotide having the nucleic acid
sequence of SEQ ID NO: 75, 76, 81, 82, 87, 88, 93, 94, 99, 100,
105, 106, 111, 112, 117, 118, 123, 124, 129, 130, 135, 136, 141,
142, 147, 148, 153, 154, 159, 160, 165, 166, 171, 172, 177, 178,
183, 184, 189, 190, 195, 196, 201, 202, 207, 208, 213, 214, 219,
220, 225, 226, 231, 232, 237, 238, 243, 244, 249, 250, 255, 256,
261, 262, 267, 268, 273, 274, 279, 280, 285, 286, 291, 292, 297,
298, 303, 304, 309, 310, 315, 316, 321, 322, 329, 330, 333, 334,
337, 338, 341, 342, 345, 346, 349, 350, 353, 354, 357, 358, 361,
362, 365, 366, 369, 370, 373, 374, 377, 378, 381, 382, 385, 386,
389, 390, 393, 394, 397 or 398; (b) a polynucleotide encoding a
polypeptide having the amino acid sequence of SEQ ID NO: 324, 326,
328, 401 or 403; (c) a polynucleotide capable of hybridizing to a
MRP-1 gene, wherein said polynucleotide is having a substitution or
deletion of at least one nucleotide at a position corresponding to
position 124667 of the MRP-1 gene (Accession No: AC026452), 1884,
1720 to 1723, 1163, 926, 437, 381, 233, 189, 440 or 1625 of the
MRP-1 gene (Accession No: U07050), 39508 of the MRP-1 gene (GI No:
7209451), 79, 88 or 249 of the MRP-1 gene (Accession No: AF022830),
95 or 259 of the MRP-1 gene (Accession No: AF022831), 57998, 57853
or 53282 of the MRP-1 gene (GI No: 7209451), 137710, 137667, 38646
or 137647 of the MRP-1 gene (Accession No: AC026452), 27159, 27258,
34206 to 34207, 34218, 34215, 55156 or 55472 of the MRP-1 gene
(Accession No: AC003026), 14008, 17970, 18195, 21133, 18067, 17900
of the MRP-1 gene (Accession No: U91318), or 150727 or 33551 of the
MRP-1 gene (Accession No: AC025277), 174 of the MRP-1 gene
(Accession No: AF022828), 248 or 258 of the MRP-1 gene (Accession
No: AF022829), 51798 or 50892 of the MRP-1 gene (Accession No: GI
3582311), 37971 of the MRP-1 gene (Accession No: GI 7363401),
55296, 55132, 55114, 55112 or 20097 to 20099 of the MRP-1 gene
(Accession No: GI 2815549), 109 to 122, 76 to 78, 73 to 78, 70 to
78, 67 to 78 or 58 to 78 of the MRP-1 gene (Accession No: GI
4826837), 60357, 61786 or 39541 of the MRP-1 gene (Accession No: GI
7209451) or a insertion of at least one nucleotide at a position
corresponding to position 55156/55157 of the MRP-1 gene (Accession
No: AC003026) or 437/438 or 926/927 of the MRP-1 gene (Accession
No: U07050) or 76437/76438 of the MRP-1 gene (Accession No: GI
7209451); (d) a polynucleotide capable of hybridizing to a MRP-1
gene, wherein said polynucleotide is having at a position
corresponding to position 124667 of the MRP-1 gene (Accession No:
AC026452) a C, at a position corresponding to position 1884 of the
MRP-1 gene (Accession No: U07050) a A, at a position corresponding
to position 1720 to 1723 of the MRP-1 gene (Accession No: U07050) a
deletion, at a position corresponding to position 1163 of the MRP-1
gene (Accession No: U07050) a T, at a position corresponding to
position 926/927 of the MRP-1 gene (Accession No: U07050) a
insertion, at a position corresponding to position 437/438 of the
MRP-1 gene (Accession No: U07050) a insertion, at a position
corresponding to position 381 of the MRP-1 gene (Accession No:
U07050) a G, at a position corresponding to position 233 of the
MRP-1 gene (Accession No: U07050) an A, at a position corresponding
to position 189 of the MRP-1 gene (Accession No: U07050) an A, at a
position corresponding to position 39508 of the MRP-1 gene (GI No:
7209451) an A, at a position corresponding to position 174 of the
MRP-1 gene (Accession No: AF022828) a T, at a position
corresponding to position 248 of the MRP-1 gene (Accession No:
AF022829) an A, at a position corresponding to position 258 of the
MRP-1 gene (Accession No: AF022829) a G, at a position
corresponding to position 79 of the MRP-1 gene (Accession No:
AF022830) an A, at a position corresponding to position 88 of the
MRP-1 gene (Accession No: AF022830) a C, at a position
corresponding to position 249 of the MRP-1 gene (Accession No:
AF022830) a G, at a position corresponding to position 95 of the
MRP-1 gene (Accession No: AF022831) a C, at a position
corresponding to position 259 of the MRP-1 gene (Accession No:
AF022831) a G, at a position corresponding to position 57998 of the
MRP-1 gene (GI No: 7209451) a T, at a position corresponding to
position 57853 of the MRP-1 gene (GI No: 7209451) a T, at a
position corresponding to position 53282 of the MRP-1 gene (GI No:
7209451) a G, at a position corresponding to position 137710 of the
MRP-1 gene (Accession No: AC026452) a G, at a position
corresponding to position 137667 of the MRP-1 gene (Accession No:
AC026452) a T, at a position corresponding to position 137647 of
the MRP-1 gene (Accession No: AC026452) a T, at a position
corresponding to position 27159 of the MRP-1 gene (Accession No:
AC003026) a C, at a position corresponding to position 27258 of the
MRP-1 gene (Accession No: AC003026) an A, at a position
corresponding to position 34206 to 34207 of the MRP-1 gene
(Accession No: AC003026) a deletion, at a position corresponding to
position 34215 of the MRP-1 gene (Accession No: AC003026) a C, at a
position corresponding to position 55156/55157 of the MRP-1 gene
(Accession No: AC003026) a insertion, at a position corresponding
to position 55472 of the MRP-1 gene (Accession No: AC003026) a C,
at a position corresponding to position 14008 of the MRP-1 gene
(Accession No: U91318) an A, at a position corresponding to
position 150727 of the MRP-1 gene (Accession No: AC025277) an A, at
a position corresponding to position 17970 of the MRP-1 gene
(Accession No: U91318) a deletion, at a position corresponding to
position 18195 of the MRP-1 gene (Accession No: U91318) an A, at a
position corresponding to position 21133 of the MRP-1 gene
(Accession No: U91318) an A, at a position corresponding to
position 34218 of the MRP-1 gene (Accession No: AC003026) an A, at
a position corresponding to position 18067 of the MRP-1 gene
(Accession No: U91318) a T, at a position corresponding to position
440 of the MRP-1 gene (Accession No: U07050) a T, at a position
corresponding to position 1625 of the MRP-1 gene (Accession No:
U07050) an A, at a position corresponding to position 17900 of the
MRP-1 gene (Accession No: U91318) a T, at a position corresponding
to position 38646 of the MRP-1 gene (Accession No: AC026452) a C,
at a position corresponding to position 33551 of the MRP-1 gene
(Accession No: AC025277) an A, at a position corresponding to
position 51798 of the MRP-1 gene (Accession No: 3582311) an G, at a
position corresponding to position 37971 of the MRP-1 gene
(Accession No: 7363401) an A, at a position corresponding to
position 50892 of the MRP-1 gene (Accession No: 3582311) an A, at a
position corresponding to position 55296 of the MRP-1 gene
(Accession No: 2815549) an A, at a position corresponding to
position 55132 of the MRP-1 gene (Accession No: 2815549) an A, at a
position corresponding to position 55114 of the MRP-1 gene
(Accession No: 2815549) an G, at a position corresponding to
position 55112 of the MRP-1 gene (Accession No: 2815549) an G, at a
position corresponding to position 109 to 122 of the MRP-1 gene
(Accession No: 4826837) deletions, at a position corresponding to
position 76 to 78 of the MRP-1 gene (Accession No: 4826837)
deletions, at a position corresponding to position 73 to 78 of the
MRP-1 gene (Accession No: 4826837) deletions, at a position
corresponding to position 70 to 78 of the MRP-1 gene (Accession No:
4826837) deletions, at a position corresponding to position 67 to
78 of the MRP-1 gene (Accession No: 4826837) deletions, at a
position corresponding to position 58 to 78 of the MRP-1 gene
(Accession No: 4826837) deletions, at a position corresponding to
position 20097 to 20099 of the MRP-1 gene (Accession No: 2815549)
deletions, at a position corresponding to position 60357 of the
MRP-1 gene (Accession No: 7209451) a T, at a position corresponding
to position 61786 of the MRP-1 gene (Accession No: 7209451) an A,
at a position corresponding to position 76437/76438 of the MRP-1
gene (Accession No: 7209451) an insertion or at a position
corresponding to position 39541 of the MRP-1 gene (Accession No:
7209451) an A; (e) a polynucleotide encoding an MRP-1 polypeptide
or fragment thereof, wherein said polypeptide comprises an amino
acid substitution at position 329, 433 or 723 of the MRP-1
polypeptide (Accession No: P33527) or 73 or 989 of the MRP-1
polypeptide (Accession No: GI 2828206); and (f) a polynucleotide
encoding an MRP-1 polypeptide or fragment thereof, wherein said
polypeptide comprises an amino acid substitution of Phe to Cys at
position 329, Arg to Ser at position 433 or Arg to Gin at position
723 of the MRP-1 polypeptide (Accession No: P33527) or Thr to lie
at position 73 or Ala to Thr at position 989 of the MRP-1
polypeptide (Accession No: GI 2828206).
2. A polynucleotide of claim 1, wherein said polynucleotide is
associated with a disease selected from the group consisting of
cancer diseases and multidrug resistance related diseases.
3. A polynucleotide of any one of claims 1 or 2 which is DNA or
RNA.
4. A gene comprising the polynucleotide of any one of claims 1 or
2.
5. The gene of claim 4, wherein a nucleotide deletion, addition
and/or substitution results in altered expression of the variant
gene compared to the corresponding wild type gene.
6. A vector comprising a polynucleotide of any one of claims 1 to 3
or the gene of claim 4 or 5.
7. The vector of claim 6, wherein the polynucleotide is operatively
linked to expression control sequences allowing expression in
prokaryotic or eukaryotic cells or isolated fractions thereof.
8. A host cell genetically engineered with the polynucleotide of
any one of claims 1 to 3, the gene of claim 4 or 5 or the vector of
claim 6 or 7.
9. A method for producing a molecular variant MRP-1 polypeptide or
fragment thereof comprising (a) culturing the host cell of claim 8;
and (b) recovering said protein or fragment from the culture.
10. A method for producing cells capable of expressing a molecular
variant MRP-1 polypeptide comprising genetically engineering cells
with the polynucleotide of any one of claims 1 to 3, the gene of
claim 4 or 5 or the vector of claim 6 or 7.
11. A polypeptide or fragment thereof encoded by the polynucleotide
of any one of claims 1 to 3, the gene of claim 4 or 5 or obtainable
by the method of claim 9 or from cells produced by the method of
claim 10.
12. An antibody which binds specifically to the polypeptide of
claim 11.
13. The antibody of claim 12 which specifically recognizes an
epitope containing one or more amino acid substitution(s) resulting
from a nucleotide exchange as defined in claim 1 or 5.
14. The antibody of claim 12 or 13 which is monoclonal or
polyclonal.
15. A transgenic non-human animal comprising at least one
polynucleotide of any one of claims 1 to 3, the gene of claim 4 or
5 or the vector of claim 6 or 7.
16. The transgenic non-human animal of claim 15 which is a mouse, a
rat or a zebrafish.
17. A solid support comprising one or a plurality of the
polynucleotide of any one of claims 1 to 3, the gene of claim 4 or
5, the vector of claim 6 or 7, the polypeptide of claim 11, the
antibody of claim 12 or 13 or the host cell of claim 8 in
immobilized form.
18. The solid support of claim 17, wherein said solid support is a
membrane, a glass- or polypropylene- or silicon-chip, are
oligonucleotide-conjugated beads or a bead array, which is
assembled on an optical filter substrate.
19. An in vitro method for identifying a single nucleotide
polymorphism said method comprising the steps of: (a) isolating a
polynucleotide of any one claims 1 to 3 or the gene of claim 4 or 5
from a plurality of subgroups of individuals, wherein one subgroup
has no prevalence for a MRP-1 associated disease and at least one
or more further subgroup(s) do have prevalence for a MRP-1
associated disease; and (b) identifying a single nucleotide
polymorphism by comparing the nucleic acid sequence of said
polynucleotide or said gene of said one subgroup having no
prevalence for a MRP-1 associated disease with said at least one or
more further subgroup(s) having a prevalence for a MRP-1 associated
disease.
20. A method for identifying and obtaining a pro-drug or a drug
capable of modulating the activity of a molecular variant of a
MRP-1 polypeptide comprising the steps of: (a) contacting the
polypeptide of claim 11, the solid support of claim 17 or 18, a
cell expressing a molecular variant gene comprising a
polynucleotide of any one of claims 1 to 3, the gene of claim 4 or
5 or the vector of claim 6 or 7 in the presence of components
capable of providing a detectable signal in response to drug
activity with a compound to be screened for pro-drug or drug
activity; and (b) detecting the presence or absence of a signal or
increase or decrease of a signal generated from the pro-drug or the
drug activity, wherein the absence, presence, increase or decrease
of the signal is indicative for a putative pro-drug or drug.
21. A method for identifying and obtaining an inhibitor of the
activity of a molecular variant of a MRP-1 polypeptide comprising
the steps of: (a) contacting the protein of claim 11, the solid
support of claim 17 or 18 or a cell expressing a molecular variant
gene comprising a polynucleotide of any one of claims 1 to 3 or the
gene of claim 4 or 5 or the vector of claim 6 or 7 in the presence
of components capable of providing a detectable signal in response
to drug activity with a compound to be screened for inhibiting
activity; and (b) detecting the presence or absence of a signal or
increase or decrease of a signal generated from the inhibiting
activity, wherein the absence or decrease of the signal is
indicative for a putative inhibitor.
22. The method of claim 20 or 21, wherein said cell is a cell of
claim 8, obtained by the method of claim 10 or can be obtained by
the transgenic non-human animal of claim 15 or 16.
23. A method of identifying and obtaining a pro-drug or drug
capable of modulating the activity of a molecular variant of a
MRP-1 polypeptide comprising the steps of: (a) contacting the host
cell of claim 8, the cell obtained by the method of claim 10, the
polypeptide of claim 11 or the solid support of claim 17 or 18 with
the first molecule known to be bound by a MRP-1 polypeptide to form
a first complex of said polypeptide and said first molecule; (b)
contacting said first complex with a compound to be screened, and
(c) measuring whether said compound displaces said first molecule
from said first complex.
24. A method of identifying and obtaining an inhibitor capable of
modulating the activity of a molecular variant of a MRP-1
polypeptide or its gene product comprising the steps of: (a)
contacting the host cell of claim 8, the cell obtained by the
method of claim 10, the protein of claim 11 or the solid support of
claim 17 or 18 with the first molecule known to be bound by a MRP-1
polypeptide to form a first complex of said polypeptide and said
first molecule; (b) contacting said first complex with a compound
to be screened, and (c) measuring whether said compound displaces
said first molecule from said first complex.
25. The method of claim 23 or 24, wherein said measuring step
comprises measuring the formation of a second complex of said
polypeptide and said compound.
26. The method of any one of claims 23 to 25, wherein said
measuring step comprises measuring the amount of said first
molecule that is not bound to said polypeptide.
27. The method of any one of claims 23 to 26, wherein said first
molecule is labeled.
28. A method for the production of a pharmaceutical composition
comprising the steps of the method of any one of claims 20 to 27;
and the further step of formulating the compound identified and
obtained or a derivative thereof in a pharmaceutically acceptable
form.
29. A method of diagnosing a disorder related to the presence of a
molecular variant of a MRP-1 gene or susceptibility to such a
disorder comprising determining the presence of a polynucleotide of
any one of claims 1 to 3 or the gene of claim 4 or 5 in a sample
from a subject.
30. The method of claim 29 further comprising determining the
presence of a polypeptide of claim 11 or the antibody of any one of
claims 12 to 14.
31. A method of diagnosing a disorder related to the presence of a
molecular variant of a MRP-1 gene or susceptibility to such a
disorder comprising determining the presence of a polypeptide of
claim 11 or the antibody of any one of claims 12 to 14 in a sample
from a subject.
32. The method of any one of claims 29 to 31, wherein said disorder
is a cancer disease or a disease related to multidrug
resistance.
33. The method of any one of claims 29 to 32 comprising PCR, ligase
chain reaction, restriction digestion, direct sequencing, nucleic
acid amplification techniques, hybridization techniques or
immunoassays.
34. A method of detection of the polynucleotide of any one of
claims 1 to 3 or the gene of claim 4 or 5 in a sample comprising
the steps of (a) contacting the solid support of claim 17 or 18
with the sample under conditions allowing interaction of the
polynucleotide of claim 1 to 3 or the gene of claim 4 or 5 with the
immobilized targets on a solid support and; (b) determining the
binding of said polynucleotide or said gene to said immobilized
targets on a solid support.
35. An in vitro method for diagnosing a disease comprising the
steps of the method of claim 34, wherein binding of said
polynucleotide or gene to said immobilized targets on said solid
support is indicative for the presence or the absence of said
disease or a prevalence for said disease.
36. A diagnostic composition comprising the polynucleotide of any
one of claims 1 to 3, the gene of claim 4 to 5, the vector of claim
6 or 7, the polypeptide of claim 11 or the antibody of any one of
the claims 12 to 14.
37. A pharmaceutical composition comprising the polynucleotide of
any one of claims 1 to 3, the gene of claim 4 or 5, the vector of
claim 6 or 7, the polypeptide of claim 11 or the antibody of any of
the claims 12 to 14.
38. Use of the polynucleotide of any one of claims 1 to 3, the gene
of claim 4 or 5, the vector of claim 6 or 7, the polypeptide of
claim 11, the polynucleotides having at a position corresponding to
position 926 of the MRP-1 gene (Accession No: U07050) a T
insertion, at a position corresponding to position 79 of the MRP-1
gene (Accession No: AF022830) an A or at a position corresponding
to position 137647 of the MRP-1 gene (Accession No: AC026452) a T,
or at a position corresponding to position 150727 of the MRP-1 gene
(Accession No: AC025277) an A, or the antibody of any of the claims
12 to 14 for the preparation of a diagnostic composition for
diagnosing a disease.
39. Use of the polynucleotide of any one of claims 1 to 3, the gene
of claim 4 or 5, the vector of claim 6 or 7, the polypeptide of
claim 11, the polynucleotides having at a position corresponding to
position 926 of the MRP-1 gene (Accession No: U07050) a T
insertion, at a position corresponding to position 79 of the MRP-1
gene (Accession No: AF022830) an A or at a position corresponding
to position 137647 of the MRP-1 gene (Accession No: AC026452) a T,
or at a position corresponding to position 150727 of the MRP-1 gene
(Accession No: AC025277) an A, or the antibody of any of the claims
12 to 14 for the preparation of a pharmaceutical composition for
treating a disease.
40. The use of claim 38 or 39, wherein said disease is cancer or a
disease related to multidrug resistance.
41. The use of claim 40 or the polynucleotide of claim 2, wherein
said cancer disease is renal cancer.
42. A diagnostic kit for detection of a single nucleotide
polymorphism comprising the polynucleotide of any one of claims 1
to 3, the gene of claim 4 or 5, the vector of claim 6 or 7, the
polypeptide of claim 11, the antibody of any of the claims 12 to
14, the host cell of claim 8, the transgenic non-human animal of
claim 15 or 16 or the solid support of claim 17 or 18.
Description
[0001] The present invention relates to a polymorphic MRP-1
polynucleotide. Moreover, the invention relates to genes or vectors
comprising the polynucleotides of the invention and to a host cell
genetically engineered with the polynucleotide or gene of the
invention. Further, the invention relates to methods for producing
molecular variant polypeptides or fragments thereof, methods for
producing cells capable of expressing a molecular variant
polypeptide and to a polypeptide or fragment thereof encoded by the
polynucleotide or the gene of the invention or which is obtainable
by the method or from the cells produced by the method of the
invention. Furthermore, the invention relates to an antibody which
binds specifically the polypeptide of the invention. Moreover, the
invention relates to a transgenic non-human animal. The invention
also relates to a solid support comprising one or a plurality of
the above mentioned polynucleotides, genes, vectors, polypeptides,
antibodies or host cells. Furthermore, methods of identifying a
polymorphism, identifying and obtaining a pro-drug or drug or an
inhibitor are also encompassed by the present invention. In
addition, the invention relates to methods for producing of a
pharmaceutical composition and to methods of diagnosing a disease.
Further, the invention relates to a method of detection of the
polynucleotide of the invention. Furthermore, comprised by the
present invention are a diagnostic and a pharmaceutical
composition. Even more, the invention relates to uses of the
polynucleotides, genes, vectors, polypeptides or antibodies of the
invention. Finally, the invention relates to a diagnostic kit.
[0002] The human multidrug resistance-associated protein (MRP)
family, a subfamily of the ATP-binding cassette (ABC) protein
superfamily, currently contains seven members.. ABC proteins are
composed of transmembrane domains (TMD's), and nucleotide binding
domains (NBD's, or ATP-binding cassettes). The ability of several
of these membrane proteins to transport a wide range of anticancer
drugs out of cells and their expression in many tumor types, make
them to possible candidates involved in unexplained cases of drug
resistance (Borst et al. 2000, J Natl Cancer Inst 92 (16):
1295-1302; Borst et al. 1999, Biochimica et Biophysica Acta 1461:
347-357; Klein et al. 1999, Biochimica et Biophysica Acta 1461:
237-262).
[0003] One member of the human MRP family is MRP-1. The gene spans
at least 200 kb and contains 31 exons. Several alternatively
spliced variants of the MRP-1 mRNA could be characterized. The
MRP-1 gene, encodes an integral membrane protein of 190 kDa whose
function is the energy dependent export of substances from the
inside of cells, and from membranes, to the outside. In contrast to
P-glycoprotein that is invariably located in the apical membrane of
epithelial cells, MRP-1 is located basolaterally and, therefore,
tends to pump drugs into the body. The protein is present in many
normal tissues and occurs mainly in lung, testis and muscle and
very low in liver. The MRP-1 protein is located in plasma membranes
in different tissues, like kidney and liver (Grant et al. 1997,
Genomics 45: 368-378; Klein et al. 1999, Biochimica et Biophysica
Acta 1461, 237-262; Cole and Deeley 1998, BioEssays 20: 931-940;
Borst et al. 1999, Biochimica et Biophysica Acta 1461: 347-357). In
addition it could be shown, that beside P-glycoprotein likewise
MRP-1 is expressed in the epithelia of the choroid plexus (CP), in
which the blood-cerebrospinal-fluid (CSF) drug permeability barrier
is localized. The function of this blood-brain barrier is to
isolate the brain from circulating drugs, toxins and xenobiotics.
MRP-1 contributes to the basolateral broad-specificity
drug-permeation barrier in CP (Rao et al. 1999, Proc. Natl. Acad.
Sci. USA 96: 3900-3905).
[0004] In contrast to P-glycoprotein and to other members of the
MRP family (MRP-4 and MRP-5), e.g. MRP-2 and MRP-1 possesses an
additional N-terminal transmembrane domain (TMD0). Thus, these
proteins contain two characteristic hydrophilic, cytosolic
ATP-binding domains (NBD's) and 3 hydrophobic transmembrane
domains, which include totally 17 transmembrane segments. This is
designated as TMD0(TMD-ABC)2 arrangement (Klein et al. 1999,
Biochimica et Biophysica Acta 1461: 237-262). The NBD's are
characterized by two sequence motifs, designated "Walker A" and
"Walker B". Mutational analysis of a number of ABC proteins
indicates that these two regions are critical for ATPase function
(Walker et al. 1982, EMBO J. 1: 945-951; Schneider et al. 1998,
FEMS Microbiol. Rev. 22: 1-20). Within the Walker A motif there
exists a conserved lysine residue (GX.sub.4GKS/T), which is
essential in both nucleotide binding domains for full transport
function. This is consistent with the role of this consensus
sequence as the amino acid acceptor site of the phosphoryl moiety
of the nucleotide. In addition, ABC transporters possess a
characteristic conserved "active transport family" signature (or
"C") motif encompassing 14 amino acids (LSSGGQX.sub.3RHydXHydA).
This region is located between the Walker A and B motifs. A
possible significance of this motif referring to the binding and
hydrolysis of nucleotide could be deduced from the observation,
that it is highly conserved in NBD1, but not in NBD2 of the
MRP-related proteins. This is in contrast to observations, which
point to a invariant nature of this motif in NBD1 and NBD2 in
P-glycoproteins (Cole and Deeley 1998, BioEssays 20: 931-940).
[0005] MRP-1 and the other members of the MRP family all contain a
highly conserved "deletion" of 13 amino acids located between the
Walker A and B motifs in NBD1, which alters the spacing between the
two Walker motifs in the first nucleotide binding domain. Recent
studies have shown, that this deletion affects the folding and
activity of this domain (Hipfner et al. 1999, J. Biol. Chem. 274
(22): 15420-6). In contrast to the NBD's, the transmembrane domains
of the ABC transporters are highly divergent. This sequence
divergence is consistent with the notion that the transmembrane
domains are important determinants of the different substrate
specificities of various ABC transporters (Ueda et al. 1997, Semin.
Cancer Biol. 8 (3): 151-159; Hrycyna et al. 1998, J. Biol. Chem.
273 (27): 16631-4). The study of post-translational modification of
the MRP-1 protein by limited proteolysis and site-directed
mutagenesis revealed, that the protein is glycosylated at Asn 19
and Asn 23 in the NH2-terminal transmembrane domain and at Asn 1006
in the COOH-proximal transmembrane domain (Hipfner et al. 1997, J.
Biol. Chem. 272 (38): 23623-30). Interestingly, recent studies of
deletion mutants of MRP-1, by the removal of the full TMD0 region,
indicated that this region is neither required for the transport
function of MRP-1 nor for its proper routing to the lateral plasma
membrane compartment (Bakos et al. 1998, J.Biol.Chem. 273:
32167-32175).
[0006] The members of the MRP family transport anionic drugs, like
methotrexate, neutral drugs conjugated to acidic ligands, such as
glutathione (GSH), glucuronate, or sulfate. While for MRP-2 the
major physiologic function is the transport of bilirubin
glucuronides and other organic anions from liver into bile, for
MRP-1 it is the transport of the cysteinyl leukotriene LTC.sub.4.
This is an important chemical mediator of inflammatory responses in
receptor-mediated, signal transduction pathways that control
vascular permeability and smooth muscle contraction. So far no
major physiologic function is known for the other members of the
MRP family. MRP-1,-2 and -3 can additionally cause resistance to
neutral organic drugs that are not known to be conjugated to acidic
ligands by transporting these drugs together with free GSH (Borst
et al. 2000, J Natl Cancer Inst 92 (16): 1295-1302; Hipfner et al.
1999, Biochimica et Biophysica Acta 1461: 359-376). Although MRP-1,
MRP-2 and MRP-3 have many common substrates, the three transport
proteins may differ in their relative affinities for individual
compounds. LTC.sub.4 remains the highest affinity substrate known
for MRP-1. In addition to the cysteinyl leukotriene LTC.sub.4 many
of the identified endogenous MRP-1 substrates, like glutathione
disulfide (GSSG) or bilirubin glucuronides are well characterized
MRP-2 substrates (Heijn et al. 1997, Biochim. Biophys. Acta 1326:
12-22; Jedlitschky et al. 1997, Biochem. J. 327: 305-310). Beside
LTC.sub.4 the preferred substrates of MRP-1 are organic anions,
like drugs conjugated to glutathione (GSH), glucuronate, or
sulfate. MRP-1 transports for example substrates, such as
methotrexate (MTX) or arsenite (H.sub.3AsO.sub.3). Likewise a
variety of other GSH-conjugated xenobiotics, including conjugates
of the activated forms of the potent carcinogen aflatoxin B1 can be
actively transported by MRP-1, suggesting a protective role of
MRP-1 in chemical carcinogenesis (Loe et al. 1997, Mol. Pharmacol.
51 (6): 1034-41). In contrast to that, P-glycoprotein has a low
affinity for such negatively charged compounds.
[0007] Glutathione conjugation by GSTs and transport of glutathione
S-conjugates out of cells into the extracellular space by MRP-1
have been shown to work as a system in the detoxification of many
xenobiotics among them many anticancer drugs (Zhang et al., 1998,
Int J Onc 12: 871-882). Because of that, the degree of expression
and the functionality of the MRP-1 gene product can affect the
therapeutic effectiveness of such agents. This is of particular
importance in cancer therapy where high MRP-1, as well as P-gp
expression and activity correlate with the resistance of cancer
cells against chemotherapeutic drugs (Gottesman et al. 1996, Curr.
Biol. 6: 610-617; Nooter and Stoter 1996, Path. Res. Pract. 192:
768-780).
[0008] Utilization of chemotherapy for the treatment of tumors can
be limited by its hematological toxicity. Transduction of
hematopoietic progenitors with the multidrug resistance 1 (MDR-1)
or with the MRP-1 gene should provide protection from toxic effects
of chemotherapeutic agents. The interest in the use of MRP-1 as an
alternative to MDR-1 for bone marrow protection lies in its
different modulation.
[0009] Because MRP-1 expression is not reversed by agents, that
decrease MDR-1 tumor resistance, these reversal agents can be used
without reversing bone marrow (BM) protection of the MRP-1
transduced hematopoietic cells. These transduced cells have shown
increased resistance to doxorubicin, vincristine and etoposide. In
mice, a retrovirus-mediated MRP-1 gene transfer into hematopoietic
cells leads to a protection from chemotherapy-induced leukopenia
(Machiels et al. 1999, Hum Gene Ther 10 (5): 801-11; D'Hondt et al.
1997, Hum Gene Ther 8 (15): 1745-51).
[0010] For understanding the physiological mechanisms of action of
MRP-1, such as mechanisms by which MRP-1 transports compounds and
mediates multidrug resistance, mrp-1 knockout models in vitro, as
well as in vivo have been generated (Wijnholds et al. 1997, Nat Med
3: 1275-1279). Because both the human and murine MRP-1 have an 88%
amino acid identity and both can induce multidrug resistance when
their respective cDNA's are transfected into drug-sensitive cells,
it is conceivable that results from knockout studies can be
transferred to humans (Stride et al. 1997, Mol Pharmacol 52:
344-353). A total block of the murine mrp-1 has been found to be
compatible with life, suggesting that MRP-1 inhibitors can be
safely used for treating cancer patients. The studies with mrp-1
knockout mice have given detailed insights in the MRP-1 transport
characteristics, so that this protein catalyzes both the export of
certain glutathione-S-conjugates and a cotransport of GSH and drugs
or endogenous metabolites (Rappa et al. 1999, Biochem Pharmacol 58:
557-562).
[0011] Different forms of multidrug resistance (MDR) have been
characterized. The classical MDR is defined by overexpression of
P-glycoprotein, while the non-Pgp MDR phenotype has typically no
expression of P-glycoprotein, but is caused by an overexpression of
MRP-1. Such an overexpression has been observed so far in
multidrug-resistant cell lines derived from many different tissue
and tumor types, including both small cell and large cell lung
cancer, carcinomas of the colon, breast, bladder, prostate, thyroid
and cervix, glioma, neuroblastoma, fibrosarcoma, and various forms
of leukemia (Hipfner et al. 1999, Biochimica et Biophysica Acta
1461: 359-376). Furthermore a cell line from renal cell carcinoma
(RCC) could be established, which show resistance to adriamycin and
epirubicin, in addition the cells demonstrated cross-resistance to
cisplatin and 5-fluororubicin. Beside elevated MDR-1, GST-pi and
topoisomerase II mRNA levels, likewise the mRNA content for MRP-1
was higher than in a control cell line (Yu et al. 2000, Urol. Res.
28 (2): 86-92).
[0012] Multidrug resistance caused by MRP-1 and P-gp is
characterized by an ATP-dependent reduction in drug accumulation.
In respect to the drug resistance profiles of transfected cells,
which overexpress P-gp or MRP-1 it could be shown that the
substrate specificity of MRP-1 and P-glycoprotein is similar.
[0013] MRP-1 transfected mammalian cells are resistant to
anthracyclines, such as doxorubicin and daunorubicin, to vinca
alkaloids, such as vincristine and to the etoposide VP-16. The
transfected cells accumulate lower levels of these drugs than do
control cells (Zhu et al. 1997, Oncol. Res. 9: 229-236). In
addition resistance to the vinca alkaloid vinblastine, to
colchicine and to the taxane paclitaxel have been observed, but to
a rather lower extent in MRP-1 transfected cells than in P-gp
overexpressing cells. The basis of this differential sensitivity is
still unknown. MRP-1 also confers resistance to certain antimonial
and arsenical oxyanions (Cole et al. 1994, Cancer Res. 54:
5902-10).
[0014] Considerable interest exists in elucidating the potential
involvement of MRP-1 in clinical MDR. For the analysis of the MRP-1
expression levels and its localization within both normal and
malignant tissues, a number of different MRP-1 antibodies have been
used in immunoassays (Flens et al. 1994, Cancer Res. 54 (17):
4557-63; Hipfner et al. 1994, Cancer Res. 54 (22): 5788-92). The
expression of the MRP1 protein and/or mRNA has been detected in
almost every tumor type examined. In the following some examples of
the tumor types, which were analyzed: solid tumors, such as lung
tumors, neuroblastoma, melanoma, retinoblastoma, breast and
prostate cancer, as well as hematological malignancies (Takebayashi
et al. 1998, Cancer 82 (4): 661-666; Campling et al. 1997, Clin.
Cancer Res. 3 (1): 115-22; Sullivan et al. 1998, Clin. Cancer Res.
4 (6): 1393-1403; Filipits et al. 1997, Leukemia 11 (7): 1073-7).
Among the common tumor types, expression of high levels of MRP1 is
particularly frequent in the major histologic forms of non-small
cell lung cancer. These studies suggest that MRP1 may be involved
in multidrug resistance of some tumor types or subgroups of
patients, but up to now no comprehensive picture of the general
revelance of this protein to clinical multidrug resistance has
defined (Hipfner et al. 1999, Biochimica et Biophysica Acta 1461:
359-376).
[0015] Nevertheless several studies have detected MRP-1 expression
levels to be of prognostic significance. In childhood neuroblastoma
it could be shown, that the amplification of the N-myc oncogene is
a powerful indicator of poor response to chemotherapy and poor
outcome. The analysis of neuroblastoma tumor samples revealed
significantly higher MRP-1 mRNA levels in tumors with N-myc
amplification, than in tumors without such an amplification. In
addition a correlation between levels of MRP-1 mRNA and a reduced
survival rate independent of the N-myc amplification could be found
(Norris et al. 1996, N. Engl. J. Med. 334 (4): 231-8).
[0016] The potential role of drug transporters in clinical
multidrug resistance has lead to a search for strategies, which
allow either an inhibition of these drug pumps, or a reduction of
the expression in cancer patients. In respect to MRP's the attempts
to find inhibitors have concentrated to MRP-1 and MRP-2. Examples
of potent competitive inhibitors are high affinity substrates, such
as leukotriene C4, S-decylglutathione and the leukotriene D4
anatgonist MK571. Other inhibitors are organic acids, such as
probenecid and benzobromarone, which were originally developed to
inhibit transport of uric acid (Borst et al. 2000, J Natl Cancer
Inst 92 (16): 1295-1302). Furthermore experiments using polarized
cell lines and ovarian carcinoma cells, both stably expressing
MRP-1 cDNA have revealed, that V-104 (a pipecolinate derivative)
partially inhibits daunorubicin transport by MRP-1. In addition
this agent reverses etoposide resistence of MRP-1 expressing
ovarian cancer cells (Evers et al. 2000, Br. J. Cancer 83 (3):
366-74). Another promising strategy for overcoming MRP-1 induced
multidrug resistance is to use antisense oligonucleotides against
this drug transporter. In MRP-1 transfected HeLa cells the
treatment with an antisense oligonucleotide, targeted to the coding
region region of the MRP-1 mRNA results in a greater than 90%
reduction of the MRP-1 mRNA level. Under these conditions an
increased sensitivity to doxorubicin was observed (Stewart et al.
1996, Biochem. Pharmacol. 51 (4): 461-9). The findings concerning
these two strategies have potential implications for the treatment
of drug-resistant tumors.
[0017] Thus, means and methods for diagnosing and treating a
variety of diseases and disorders based on dysfunctions or
dysregulations of drug transport were not available yet but are
nevertheless highly desirable. Thus, the technical problem
underlying the present invention is to comply with the above
specified needs.
[0018] The solution to this technical problem is achieved by
providing the embodiments characterized in the claims.
[0019] Accordingly, the present invention relates to a
polynucleotide comprising a polynucleotide selected from the group
consisting of:
[0020] (a) a polynucleotide having the nucleic acid sequence of SEQ
ID NO: 75, 76, 81, 82, 87, 88, 93, 94, 99, 100, 105, 106, 111, 112,
117, 118, 123, 124, 129, 130, 135, 136, 141, 142, 147, 148, 153,
154, 159, 160, 165, 166, 171, 172, 177, 178, 183, 184, 189, 190,
195, 196, 201, 202, 207, 208, 213, 214, 219, 220, 225, 226, 231,
232, 237, 238, 243, 244, 249, 250, 255, 256, 261, 262, 267, 268,
273, 274, 279, 280, 285, 286, 291, 292, 297, 298, 303, 304, 309,
310, 315, 316, 321, 322, 329, 330, 333, 334, 337, 338, 341, 342,
345, 346, 349, 350, 353, 354, 357, 358, 361, 362, 365, 366, 369,
370, 373, 374, 377, 378, 381, 382, 385, 386, 389, 390, 393, 394,
397 or 398;
[0021] (b) a polynucleotide encoding a polypeptide having the amino
acid sequence of SEQ ID NO: 324, 326, 328, 401 or 403;
[0022] (c) a polynucleotide capable of hybridizing to a MRP-1 gene,
wherein said polynucleotide is having a substitution or deletion of
at least one nucleotide at a position corresponding to position
124667 of the MRP-1 gene (Accession No: AC026452), 1884, 1720 to
1723, 1163, 926, 437, 381, 233, 189, 440 or 1625 of the MRP-1 gene
(Accession No: U07050), 39508 of the MRP-1 gene (GI No: 7209451),
79, 88 or 249 of the MRP-1 gene (Accession No: AF022830), 95 or 259
of the MRP-1 gene (Accession No: AF022831), 57998, 57853 or 53282
of the MRP-1 gene (GI No: 7209451), 137710, 137667, 38646 or 137647
of the MRP-1 gene (Accession No: AC026452), 27159, 27258, 34206 to
34207, 34218, 34215, 55156 or 55472 of the MRP-1 gene (Accession
No: AC003026), 14008, 17970, 18195, 21133, 18067, 17900 of the
MRP-1 gene-(Accession No: U91318), or 150727 or 33551 of the MRP-1
gene (Accession No: AC025277), 174 of the MRP-1 gene (Accession No:
AF022828), 248 or 258 of the MRP-1 gene (Accession No: AF022829),
51798 or 50892 of the MRP-1 gene (Accession No: GI 3582311), 37971
of the MRP-1 gene (Accession No: GI 7363401), 55296, 55132, 55114,
55112 or 20097 to 20099 of the MRP-1 gene (Accession No: GI
2815549), 109 to 122, 76 to 78, 73 to 78, 70 to 78, 67 to 78 or 58
to 78 of the MRP-1 gene (Accession No: GI 4826837), 60357, 61786 or
39541 of the MRP-1 gene (Accession No: GI 7209451) or a insertion
of at least one nucleotide at a position corresponding to position
55156/55157 of the MRP-1 gene (Accession No: AC003026), 437/438 or
926/927 of the MRP-1 gene (Accession No: U07050) or 76437/76438 of
the MRP-1 gene (Accession No: GI 7209451);
[0023] (d) a polynucleotide capable of hybridizing to a MRP-1 gene,
wherein said polynucleotide is having at a position corresponding
to position 124667 of the MRP-1 gene (Accession No: AC026452) a C,
at a position corresponding to position 1884 of the MRP-1 gene
(Accession No: U07050) a A, at a position corresponding to position
1720 to 1723 of the MRP-1 gene (Accession No: U07050) a deletion,
at a position corresponding to position 1163 of the MRP-1 gene
(Accession No: U07050) a T, at a position corresponding to position
926/927 of the MRP-1 gene (Accession No: U07050) a insertion, at a
position corresponding to position 437/438 of the MRP-1 gene
(Accession No: U07050) a insertion, at a position corresponding to
position 381 of the MRP-1 gene (Accession No: U07050) a G, at a
position corresponding to position 233 of the MRP-1 gene (Accession
No: U07050) an A, at a position corresponding to position 189 of
the MRP-1 gene (Accession No: U07050) an A, at a position
corresponding to position 39508 of the MRP-1 gene (GI No: 7209451)
an A, at a position corresponding to position 174 of the MRP-1 gene
(Accession No: AF022828) a T, at a position corresponding to
position 248 of the MRP-1 gene (Accession No: AF022829) an A, at a
position corresponding to position 258 of the MRP-1 gene (Accession
No: AF022829) a G, at a position corresponding to position 79 of
the MRP-1 gene (Accession No: AF022830) an A, at a position
corresponding to position 88 of the MRP-1 gene (Accession No:
AF022830) a C, at a position corresponding to position 249 of the
MRP-1 gene (Accession No: AF022830) a G, at a position
corresponding to position 95 of the MRP-1 gene (Accession No:
AF022831) a C, at a position corresponding to position 259 of the
MRP-1 gene (Accession No: AF022831) a G, at a position
corresponding to position 57998 of the MRP-1 gene (GI No: 7209451)
a T, at a position corresponding to position 57853 of the MRP-1
gene (GI No: 7209451) a T, at a position corresponding to position
53282 of the MRP-1 gene (GI No: 7209451) a G, at a position
corresponding to position 137710 of the MRP-1 gene (Accession No:
AC026452) a G, at a position corresponding to position 137667 of
the MRP-1 gene (Accession No: AC026452) a T, at a position
corresponding to position 137647 of the MRP-1 gene (Accession No:
AC026452) a T, at a position corresponding to position 27159 of the
MRP-1 gene (Accession No: AC003026) a C, at a position
corresponding to position 27258 of the MRP-1 gene (Accession No:
AC003026) an A, at a position corresponding to position 34206 to
34207 of the MRP-1 gene (Accession No: AC003026) a deletion, at a
position corresponding to position 34215 of the MRP-1 gene
(Accession No: AC003026) a C, at a position corresponding to
position 55156/55157 of the MRP-1 gene (Accession No: AC003026) a
insertion, at a position corresponding to position 55472 of the
MRP-1 gene (Accession No: AC003026) a C, at a position
corresponding to position 14008 of the MRP-1 gene (Accession No:
U91318) an A, at a position corresponding to position 150727 of the
MRP-1 gene (Accession No: AC025277) an A, at a position
corresponding to position 17970 of the MRP-1 gene (Accession No:
U91318) a deletion, at a position corresponding to position 18195
of the MRP-1 gene (Accession No: U91318) an A, at a position
corresponding to position 21133 of the MRP-1 gene (Accession No:
U91318) an A, at a position corresponding to position 34218 of the
MRP-1 gene (Accession No: AC003026) an A, at a position
corresponding to position 18067 of the MRP-1 gene (Accession No:
U91318) a T, at a position corresponding to position 440 of the
MRP-1 gene (Accession No: U07050) a T, at a position corresponding
to position 1625 of the MRP-1 gene (Accession No: U07050) an A, at
a position corresponding to position 17900 of the MRP-1 gene
(Accession No: U91318) a T, at a position corresponding to position
38646 of the MRP-1 gene (Accession No: AC026452) a C, at a position
corresponding to position 33551 of the MRP-1 gene (Accession No:
AC025277) an A, at a position corresponding to position 51798 of
the MRP-1 gene (Accession No: 3582311) an G, at a position
corresponding to position 37971 of the MRP-1 gene (Accession No:
7363401) an A, at a position corresponding to position 50892 of the
MRP-1 gene (Accession No: 3582311) an A, at a position
corresponding to position 55296 of the MRP-1 gene (Accession No:
2815549) an A, at a position corresponding to position 55132 of the
MRP-1 gene (Accession No: 2815549) an A, at a position
corresponding to position 55114 of the MRP-1 gene (Accession No:
2815549) an G, at a position corresponding to position 55112 of the
MRP-1 gene (Accession No: 2815549) an G, at a position
corresponding to position 109 to 122 of the MRP-1 gene (Accession
No: 4826837) deletions, at a position corresponding to position 76
to 78 of the MRP-1 gene (Accession No: 4826837) deletions, at a
position corresponding to position 73 to 78 of the MRP-1 gene
(Accession No: 4826837) deletions, at a position corresponding to
position 70 to 78 of the MRP-1 gene (Accession No: 4826837)
deletions, at a position corresponding to position 67 to 78 of the
MRP-1 gene (Accession No: 4826837) deletions, at a position
corresponding to position 58 to 78 of the MRP-1 gene (Accession No:
4826837) deletions, at a position corresponding to position 20097
to 20099 of the MRP-1 gene (Accession No: 2815549) deletions, at a
position corresponding to position 60357 of the MRP-1 gene
(Accession No: 7209451) a T, at a position corresponding to
position 61786 of the MRP-1 gene (Accession No: 7209451) an A, at a
position corresponding to position 76437/76438 of the MRP-1 gene
(Accession No: 7209451) an insertion or at a position corresponding
to position 39541 of the MRP-1 gene (Accession No: 7209451) an
A;
[0024] (e) a polynucleotide encoding an MRP-1 polypeptide or
fragment thereof, wherein said polypeptide comprises an amino acid
substitution at position 329, 433 or 723 of the MRP-1 polypeptide
(Accession No: P33527) or 73 or 989 of the MRP-1 polypeptide
(Accession No: GI 2828206); and
[0025] (f) a polynucleotide encoding an MRP-1 polypeptide or
fragment thereof, wherein said polypeptide comprises an amino acid
substitution of Phe to Cys at position 329, Arg to Ser at position
433 or Arg to Gln at position 723 of the MRP-1 polypeptide
(Accession No: P33527) or Thr to Ile at position 73 or Ala to Thr
at position 989 of the MRP-1 polypeptide (Accession No: GI
2828206).
[0026] In the context of the present invention the term
"polynucleotides" or the term "polypeptides" refers to different
variants of a polynucleotide or polypeptide. Said variants comprise
a reference or wild type sequence of the polynucleotides or
polypeptides of the invention as well as variants which differ
therefrom in structure or composition. Reference or wild type
sequences for the polynucleotides are Accession No: U07050,
AF022828, AF022829, AF022830, AF022831, AC026452, AC003026, U91318,
AC025277 or GI No: 7209451. Reference or wild type sequence for the
polypeptides of the invention is Accession No: P33527. The
differences in structure or composition usually occur by way of
nucleotide or amino acid substitution(s), addition(s) and/or
deletion(s). Preferred deletions in accordance with the invention
are a GGTA deletion at a position corresponding to position 1720 to
1723 of the MRP-1 gene (Accession No: U07050), an AT deletion at a
position corresponding to position 34206 to 34207 of the MRP-1 gene
(Accession No: AC003026) or a T deletion at a position
corresponding to position 17970 of the MRP-1 gene (Accession No:
U91318), preferred insertions are a TCCTTCC insertion at a position
corresponding to position 437/438 of the MRP-1 gene (Accession No:
U07050), a TGGGGC insertion at a position corresponding to position
55156/55157 of the MRP-1 gene (Accession No: AC003026) or a T
insertion at a position corresponding to position 926/927 of the
MRP-1 gene (Accession No: U07050).
[0027] Preferably, said nucleotide substitution(s), addition(s) or
deletion(s) comprised by the present invention result(s) in one or
more changes of the corresponding amino acid(s) of the polypeptides
of the invention. The variant polynucleotides and polypeptides also
comprise fragments of said polynucleotides or polypeptides of the
invention. The polynucleotides and polypeptides as well as the
aforementioned fragments thereof of the present invention are
characterized as being associated with a MRP-1 dysfunction or
dysregulation comprising, e.g., insufficient and/or altered drug
uptake. Said dysfunctions or dysregulations referred to in the
present invention cause a disease or disorder or a prevalence for
said disease or disorder. Preferably, as will be discussed below in
detail, said disease is cancer or diseases related to multidrug
resistance or any other disease caused by a dysfunction or
dysregulation due to a polynucleotide or polypeptides of the
invention, also referred to as MRP-1 gene associated diseases in
the following.
[0028] The term "hybridizing" as used herein refers to
polynucleotides which are capable of hybridizing to the
polynucleotides of the invention or parts thereof which are
associated with a MRP-1 dysfunction or dysregulation. Thus, said
hybridizing polynucleotides are also associated with said
dysfunctions and dysregulations.
[0029] Preferably, said polynucleotides capable of hybridizing to
the polynucleotides of the invention or parts thereof which are
associated with MRP-1 dysfunctions or dysregulations are at least
70%, at least 80%, at least 95% or at least 100% identical to the
polynucleotides of the invention or parts thereof which are
associated with MRP-1 dysfunctions or dysregulations. Therefore,
said polynucleotides may be useful as probes in Northern or
Southern Blot analysis of RNA or DNA preparations, respectively, or
can be used as oligonucleotide primers in PCR analysis dependent on
their respective size. Also comprised by the invention are
hybridizing polynucleotides which are useful for analysing
DNA-Protein interactions via, e.g., electrophoretic mobility shift
analysis (EMSA). Preferably, said hybridizing polynucleotides
comprise at least 10, more preferably at least 15 nucleotides in
length while a hybridizing polynucleotide of the present invention
to be used as a probe preferably comprises at least 100, more
preferably at least 200, or most preferably at least 500
nucleotides in length.
[0030] It is well known in the art how to perform hybridization
experiments with nucleic acid molecules, i.e. the person skilled in
the art knows what hybridization conditions s/he has to use in
accordance with the present invention. Such hybridization
conditions are referred to in standard text books such as Molecular
Cloning A Laboratory Manual, Cold Spring Harbor Laboratory (1989)
N.Y. Preferred in accordance with the present inventions are
polynucleotides which are capable of hybridizing to the
polynucleotides of the invention or parts thereof which are
associated with a MRP-1 dysfunction or dysregulation under
stringent hybridization conditions, i.e. which do not cross
hybridize to unrelated polynucleotides such as polynucleotides
encoding a polypeptide different from the MRP-1 polypeptides of the
invention.
[0031] The term "corresponding" as used herein means that a
position is not only determined by the number of the preceding
nucleotides and amino acids, respectively. The position of a given
nucleotide or amino acid in accordance with the present invention
which may be deleted, substituted or comprise one or more
additional nucleotide(s) may vary due to deletions or additional
nucleotides or amino acids elsewhere in the gene or the
polypeptide. Thus, under a "corresponding position" in accordance
with the present invention it is to be understood that nucleotides
or amino acids may differ in the indicated number but may still
have similar neighboring nucleotides or amino acids. Said
nucleotides or amino acids which may be exchanged, deleted or
comprise additional nucleotides or amino acids are also comprised
by the term "corresponding position". Said nucleotides or amino
acids may for instance together with their neighbors form sequences
which may be involved in the regulation of gene expression,
stability of the corresponding RNA or RNA editing, as well as
encode functional domains or motifs of the protein of the
invention.
[0032] By, e.g., "position 1720 to 1723" it is meant that said
polynucleotide comprises one or more deleted nucleotides which are
deleted between positions 1720 and position 1723 of the
corresponding wild type version of said polynucleotide. The same
applies mutatis mutandis to all other position numbers referred to
in the above embodiment which are drafted in the same format.
[0033] By, e.g., "position 437/438" it is meant that said
polynucleotide comprises one or more additional nucleotide(s) which
are inserted between positions 437 and position 438 of the
corresponding wild type version of said polynucleotide. The same
applies mutatis mutandis to all other position numbers referred to
in the above embodiment which are drafted in the same format, i.e.
two consecutive position numbers separated by a slash (/).
[0034] In accordance with the present invention, the mode and
population distribution of genetic variations in the MRP-1 gene has
been analyzed by sequence analysis of relevant regions of the human
said gene from many different individuals. It is a well known fact
that genomic DNA of individuals, which harbor the individual
genetic makeup of all genes, including the MRP-1 gene, can easily
be purified from individual blood samples. These individual DNA
samples are then used for the analysis of the sequence composition
of the alleles of the MRP-1 gene that are present in the individual
which provided the blood sample. The sequence analysis was carried
out by PCR amplification of relevant regions of said genes,
subsequent purification of the PCR products, followed by automated
DNA sequencing with established methods (e.g. ABI dyeterminator
cycle sequencing).
[0035] One important parameter that had to be considered in the
attempt to determine the individual genotypes and identify novel
variants of the MRP-1 gene by direct DNA-sequencing of PCR-products
from human blood genomic DNA is the fact that each human harbors
(usually, with very few abnormal exceptions) two gene copies of
each autosomal gene (diploidy). Because of that, great care had to
be taken in the evaluation of the sequences to be able to identify
unambiguously not only homozygous sequence variations but also
heterozygous variations. The details of the different steps in the
identification and characterization of novel polymorphisms in the
MRP-1 gene (homozygous and heterozygous) are described in the
Examples below.
[0036] Over the past 20 years, genetic heterogeneity has been
increasingly recognized as a significant source of variation in
drug response. Many scientific communications (Meyer, Ann. Rev.
Pharmacol. Toxicol. 37 (1997), 269-296 and West, J. Clin.
Pharmacol. 37 (1997), 635-648) have clearly shown that some drugs
work better or may even be highly toxic in some patients than in
others and that these variations in patient's responses to drugs
can be related to molecular basis. This "pharmacogenomic" concept
spots correlations between responses to drugs and genetic profiles
of patient's (Marshall, Nature Biotechnology, 15 (1997), 954-957;
Marshall, Nature Biotechnology, 15 (1997), 1249-1252). In this
context of population variability with regard to drug therapy,
pharmacogenomics has been proposed as a tool useful in the
identification and selection of patients which can respond to a
particular drug without side effects. This identification/selection
can be based upon molecular diagnosis of genetic polymorphisms by
genotyping DNA from leukocytes in the blood of patient, for
example, and characterization of disease (Bertz, Clin.
Pharmacokinet. 32 (1997), 210-256; Engel, J. Chromatogra. B.
Biomed. Appl. 678 (1996), 93-103). For the founders of health care,
such as health maintenance organizations in the US and government
public health services in many European countries, this
pharmacogenomics approach can represent a way of both improving
health care and reducing overheads because there is a large cost to
unnecessary drugs, ineffective drugs and drugs with side
effects.
[0037] The mutations in the variant genes of the invention sometime
result in amino acid deletion(s), insertion(s) and in particular in
substitution(s) either alone or in combination. It is of course
also possible to genetically engineer such mutations in wild type
genes or other mutant forms. Methods for introducing such
modifications in the DNA sequence of said genes are well known to
the person skilled in the art; see, e.g., Sambrook, Molecular
Cloning A Laboratory Manual, Cold Spring Harbor Laboratory (1989)
N.Y.
[0038] For the investigation of the nature of the alterations in
the amino acid sequence of the polypeptides of the invention may be
used such as BRASMOL that are obtainable from the Internet.
Furthermore, folding simulations and computer redesign of
structural motifs can be performed using other appropriate computer
programs (Olszewski, Proteins 25 (1996), 286-299; Hoffman, Comput.
Appl. Biosci. 11 (1995), 675-679). Computers can be used for the
conformational and energetic analysis of detailed protein models
(Monge, J. Mol. Biol. 247 (1995), 995-1012; Renouf, Adv. Exp. Med.
Biol. 376 (1995), 37-45). These analysis can be used for the
identification of the influence of a particular mutation on binding
and/or transport of drugs.
[0039] Usually, said amino acid deletion, addition or substitution
in the amino acid sequence of the protein encoded by the
polynucleotide of the invention is due to one or more nucleotide
substitution, insertion or deletion, or any combinations thereof.
Preferably said nucleotide substitution, insertion or deletion may
result in an amino acid substitution of F to C at position
corresponding to position 329 of the MRP-1 polypeptide (Accession
No: P33527), R to S at position corresponding to position 433 of
the MRP-1 polypeptide (Accession No: P33527) or R to Q at position
corresponding to position 723 of the MRP-1 polypeptide (Accession
No: P33527). The polypeptides of encoded by the polynucleotides of
the invention have altered biological or immunological properties
due to the mutations referred to in accordance with the present
invention. Examples for said altered properties are stability of
the polypeptides which may be effected or an altered substrate
specificity or an altered transport activity characterized by,
e.g., insufficiencies in drug transport or a complete loss of the
capability of transporting drugs.
[0040] The mutations in the MRP-1 gene detected in accordance with
the present invention are listed in Table 2. The methods of the
mutation analysis followed standard protocols and are described in
detail in the Examples. In general such methods are to be used in
accordance with the present invention for evaluating the phenotypic
spectrum as well as the overlapping clinical characteristics of
diseases or conditions related to dysfunctions or dysregulations
and diseases related to impaired drug transport. Advantageously,
the characterization of said mutants may form the basis of the
development of improved drugs, such as drugs which are used in
therapy of diseases related to multidrug resistance such as in
cancer therapy. Said methods encompass for example haplotype
analysis, single-strand conformation polymorphism analysis (SSCA),
PCR and direct sequencing. On the basis of thorough clinical
characterization of many patients the phenotypes can then be
correlated to these mutations as well as to mutations that had been
described earlier, for example in Jounaidi, Biochem Biophys Res
Commun, 221, pp. 466-470, 1996.
[0041] Also comprised by the polynucleotides referred to in the
present invention are polynucleotides which comprise at least two
of the polynucleotides specified hereinabove, i.e. polynucleotides
having a nucleotide sequence which contains at least two of the
mutations comprised by the above polynucleotides or listed in Table
2 below. This allows the study of synergistic effects of said
mutations in the MRP-1 gene and/or a polypeptide encoded by said
polynucleotide on the pharmacological profile of drugs in patients
who bear such mutant forms of the gene or similar mutant forms that
can be mimicked by the above described proteins. It is expected
that the analysis of said synergistic effects provides deeper
insights into the onset of MRP-1 dysfunctions or dysregulations or
diseases related to altered drug transport as described supra. From
said deeper insight the development of diagnostic and
pharmaceutical compositions related to MRP-1 dysfunctions or
dysregulations or diseases related to altered drug transport will
greatly benefit.
[0042] As is evident to the person skilled in the art, the genetic
knowledge deduced from the present invention can now be used to
exactly and reliably characterize the genotype of a patient.
Advantageously, diseases or a prevalence for a disease which are
associated with MRP-1 dysfunction or dysregulation, such as cancer
or other multidrug resistance related diseases referred to herein
can be predicted and preventive or therapeutical measures can be
applied accordingly. Moreover in accordance with the foregoing, in
cases where a given drug takes an unusual effect, a suitable
individual therapy can be designed based on the knowledge of the
individual genetic makeup of a subject with respect to the
polynucleotides of the invention and improved therapeutics can be
developed as will be further discussed below.
[0043] In general, the MRP-1 "status", defined by the expression
level and activity of the MRP-1 protein, can be not only altered in
many disease or disorders including cancer (see above), but can
also be variable in normal tissue, due to genetic
variations/polymorphisms. The identification of polymorphisms
associated with altered MRP-1 expression and/or activity is
important for the prediction of drug uptake and subsequently for
the prediction of therapy outcome, including side effects of
medications. Therefore, analysis of MRP-1 variations indicative of
MRP-1 function, is a valuable tool for therapy with drugs, which
are substrates of MRP-1 and has, thanks to the present invention,
now become possible.
[0044] Finally, the polynucleotides and polypeptides referred to in
accordance with the present invention are also useful as forensic
markers, which improve the identification of subjects which have
been murdered or killed by, for example a crime of violence or any
other violence and can not be identified by the well known
conventional forensic methods. The application of forensic methods
based on the detection of the polymorphisms comprised by the
polynucleotides of this invention in the genome of a subject are
particularly well suited in cases where a (dead) body is disfigured
in a severe manner such as identification by other body
characteristics such as the features of the face is not possible.
This is the case, for example, for corpses found in water which are
usually entirely disfigured. Advantageously, methods which are
based on the provision of the polynucleotides of the invention
merely require a minimal amount of tissue or cells in order to be
carried out. Said tissues or cells may be blood droplets, hair
roots, epidermal scales, salivia droplets, sperms etc. Since only
such a minimal amount of tissue or cells is required for the
identification of a subject, the polymorphism comprised by the
polynucleotides of this invention can also be used as forensic
markers in order to proof someone guilty for a crime, such as a
violation or a ravishment. Moreover, the polymorphisms comprised by
the polynucleotides of this invention can be used to proof
paternity. In accordance with the forensic methods referred herein
the presence or absence of the polynucleotides of the invention is
determined and compared with a reference sample which is
unambiguously derived from the subject to be identified. The
forensic methods which require detection of the presence or absence
of the polynucleotides of this invention in a sample of a subject
the polymorphisms comprised by the polynucleotides of this
invention can be for example PCR-based techniques which are
particularly well suited in cases where only minimal amount of
tissue or cells is available as forensic samples. On the other
hand, where enough tissue or cells is available, hybridization
based techniques may be performed in order to detect the presence
or absence of a polynucleotide of this invention. These techniques
are well known by the person skilled in the art and can be adopted
to the individual purposes referred to herein without further ado.
In conclusion, thanks to the present invention forensic means which
allow improved and reliable predictions as regards the
aforementioned aspects are now available.
[0045] In line with the foregoing, preferably, the polynucleotide
of the present invention is associated with a disease selected from
the group of cancer diseases or multidrug resistance related
diseases.
[0046] The term "cancer" used herein is very well known and
characterized in the art. Several variants of cancer exist and are
comprised by said term as meant in accordance with the invention.
For a detailed list of symptoms which are indicative for cancer it
is referred to text book knowledge, e.g. Pschyrembel.
[0047] More preferably, said cancer disease is kidney cancer, such
as renal cell carcinoma (RCC). The meaning of renal cancer is
explicitly disclosed in Example 4.
[0048] In a further embodiment the present invention relates to a
polynucleotide which is DNA or RNA.
[0049] The polynucleotide of the invention may be, e.g., DNA, cDNA,
genomic DNA, RNA or synthetically produced DNA or RNA or a
recombinantly produced chimeric nucleic acid molecule comprising
any of those polynucleotides either alone or in combination.
Preferably said polynucleotide is part of a vector, particularly
plasmids, cosmids, viruses and bacteriophages used conventionally
in genetic engineering that comprise a polynucleotide of the
invention. Such vectors may comprise further genes such as marker
genes which allow for the selection of said vector in a suitable
host cell and under suitable conditions.
[0050] The invention furthermore relates to a gene comprising the
polynucleotide of the invention.
[0051] It is well known in the art that genes comprise structural
elements which encode an amino acid sequence as well as regulatory
elements which are involved in the regulation of the expression of
said genes.. Structural elements are represented by exons which may
either encode an amino acid sequence or which may encode for RNA
which is not encoding an amino acid sequence but is nevertheless
involved in RNA function, e.g. by regulating the stability of the
RNA or the nuclear export of the RNA.
[0052] Regulatory elements of a gene may comprise promoter elements
or enhancer elements both of which could be involved in
transcriptional control of gene expression. It is very well known
in the art that a promoter is to be found upstream of the
structural elements of a gene. Regulatory elements such as enhancer
elements, however, can be found distributed over the entire locus
of a gene. Said elements could be reside, e.g., in introns, regions
of genomic DNA which separate the exons of a gene. Promoter or
enhancer elements correspond to polynucleotide fragments which are
capable of attracting or binding polypeptides involved in the
regulation of the gene comprising said promoter or enhancer
elements. For example, polypeptides involved in regulation of said
gene comprise the so called transcription factors.
[0053] Said introns may comprise further regulatory elements which
are required for proper gene expression. Introns are usually
transcribed together with the exons of a gene resulting in a
nascent RNA transcript which contains both, exon and intron
sequences. The intron encoded RNA sequences are usually removed by
a process known as RNA splicing. However, said process also
requires regulatory sequences present on a RNA transcript said
regulatory sequences may be encoded by the introns.
[0054] In addition, besides their function in transcriptional
control and control of proper RNA processing and/or stability,
regulatory elements of a gene could be also involved in the control
of genetic stability of a gene locus. Said elements control, e.g.,
recombination events or serve to maintain a certain structure of
the DNA or the arrangement of DNA in a chromosome.
[0055] Therefore, single nucleotide polymorphisms can occur in
exons of a gene which encode an amino acid sequence as discussed
supra as well as in regulatory regions which are involved in the
above discussed process. The analysis of the nucleotide sequence of
a gene locus in its entirety including, e.g., introns is in light
of the above desirable. The polymorphisms comprised by the
polynucleotides of the present invention can influence the
expression level of MRP-1 protein via mechanisms involving enhanced
or reduced transcription of the MRP-1 gene, stabilization of the
gene's RNA transcripts and alteration of the processing of the
primary RNA transcripts.
[0056] Therefore, in a furthermore preferred embodiment of the gene
of the invention a nucleotide deletion, addition and/or
substitution results in altered expression of the variant gene
compared to the corresponding wild type gene.
[0057] In another embodiment the present invention relates to a
vector comprising the polynucleotide of the invention or the gene
of the invention.
[0058] Said vector may be, for example, a phage, plasmid, viral or
retroviral vector. Retroviral vectors may be replication competent
or replication defective. In the latter case, viral propagation
generally will occur only in complementing host/cells.
[0059] The polynucleotides or genes of the invention may be joined
to a vector containing selectable markers for propagation in a
host. Generally, a plasmid vector is introduced in a precipitate
such as a calcium phosphate precipitate, or in a complex with a
charged lipid or in carbon-based clusters. Should the vector be a
virus, it may be packaged in vitro using an appropriate packaging
cell line prior to application to host cells.
[0060] In a more preferred embodiment of the vector of the
invention the polynucleotide is operatively linked to expression
control sequences allowing expression in prokaryotic or eukaryotic
cells or isolated fractions thereof.
[0061] Expression of said polynucleotide comprises transcription of
the polynucleotide, preferably into a translatable mRNA. Regulatory
elements ensuring expression in eukaryotic cells, preferably
mammalian cells, are well known to those skilled in the art. They
usually comprise regulatory sequences ensuring initiation of
transcription and optionally poly-A signals ensuring termination of
transcription and stabilization of the transcript. Additional
regulatory elements may include transcriptional as well as
translational enhancers. Possible regulatory elements permitting
expression in prokaryotic host cells comprise, e.g., the lac, trp
or tac promoter in E. coli, and examples for regulatory elements
permitting expression in eukaryotic host cells are the AOX1 or GAL1
promoter in yeast or the CMV-, SV40- , RSV-promoter (Rous sarcoma
virus), CMV-enhancer, SV40-enhancer or a globin intron in mammalian
and other animal cells. Beside elements which are responsible for
the initiation of transcription such regulatory elements may also
comprise transcription termination signals, such as the SV40-poly-A
site or the tk-poly-A site, downstream of the polynucleotide. In
this context, suitable expression vectors are known in the art such
as Okayama-Berg cDNA expression vector pcDV1 (Pharmacia), pCDM8,
pRc/CMV, pcDNA1, pcDNA3 (In-vitrogene), pSPORT1 (GIBCO BRL).
Preferably, said vector is an expression vector and/or a gene
transfer or targeting vector. Expression vectors derived from
viruses such as retroviruses, vaccinia virus, adeno-associated
virus, herpes viruses, or bovine papilloma virus, may be used for
delivery of the polynucleotides or vector of the invention into
targeted cell population. Methods which are well known to those
skilled in the art can be used to construct recombinant viral
vectors; see, for example, the techniques described in Sambrook,
Molecular Cloning A Laboratory Manual, Cold Spring Harbor
Laboratory (1989) N.Y. and Ausubel, Current Protocols in Molecular
Biology, Green Publishing Associates and Wiley Interscience, N.Y.
(1994). Alternatively, the polynucleotides and vectors of the
invention can be reconstituted into liposomes for delivery to
target cells.
[0062] The term "isolated fractions thereof" refers to fractions of
eukaryotic or prokaryotic cells or tissues which are capable of
transcribing or transcribing and translating RNA from the vector of
the invention. Said fractions comprise proteins which are required
for transcription of RNA or transcription of RNA and translation of
said RNA into a polypeptide. Said isolated fractions may be, e.g.,
nuclear and cytoplasmic fractions of eukaryotic cells such as of
reticulocytes.
[0063] The present invention furthermore relates to a host cell
genetically engineered with the polynucleotide of the invention,
the gene of the invention or the vector of the invention.
[0064] Said host cell may be a prokaryotic or eukaryotic cell; see
supra. The polynucleotide or vector of the invention which is
present in the host cell may either be integrated into the genome
of the host cell or it may be maintained extrachromosomally. In
this respect, it is also to be understood that the recombinant DNA
molecule of the invention can be used for "gene targeting" and/or
"gene replacement", for restoring a mutant gene or for creating a
mutant gene via homologous recombination; see for example Mouellic,
Proc. Natl. Acad. Sci. USA, 87 (1990), 4712-4716; Joyner, Gene
Targeting, A Practical Approach, Oxford University Press.
[0065] The host cell can be any prokaryotic or eukaryotic cell,
such as a bacterial, insect, fungal, plant, animal, mammalian or,
preferably, human cell. Preferred fungal cells are, for example,
those of the genus Saccharomyces, in particular those of the
species S. cerevisiae. The term "prokaryotic" is meant to include
all bacteria which can be transformed or transfected with a
polynucleotide for the expression of a variant polypeptide of the
invention. Prokaryotic hosts may include gram negative as well as
gram positive bacteria such as, for example, E. coli, S.
typhimurium, Serratia marcescens and Bacillus subtilis. A
polynucleotide coding for a mutant form of variant polypeptides of
the invention can be used to transform or transfect the host using
any of the techniques commonly known to those of ordinary skill in
the art. Methods for preparing fused, operably linked genes and
expressing them in bacteria or animal cells are well-known in the
art (Sambrook, supra). The genetic constructs and methods described
therein can be utilized for expression of variant polypeptides of
the invention in, e.g., prokaryotic hosts. In general, expression
vectors containing promoter sequences which facilitate the
efficient transcription of the inserted polynucleotide are used in
connection with the host. The expression vector typically contains
an origin of replication, a promoter, and a terminator, as well as
specific genes which are capable of providing phenotypic selection
of the transformed cells. The transformed prokaryotic hosts can be
grown in fermentors and cultured according to techniques known in
the art to achieve optimal cell growth. The proteins of the
invention can then be isolated from the grown medium, cellular
lysates, or cellular membrane fractions. The isolation and
purification of the microbially or otherwise expressed polypeptides
of the invention may be by any conventional means such as, for
example, preparative chromatographic separations and immunological
separations such as those involving the use of monoclonal or
polyclonal antibodies.
[0066] Thus, in a further embodiment the invention relates to a
method for producing a molecular variant MRP-1 polypeptide or
fragment thereof comprising culturing the above described host
cell; and recovering said protein or fragment from the culture.
[0067] In another embodiment the present invention relates to a
method for producing cells capable of expressing a molecular
variant MRP-1 polypeptide comprising genetically engineering cells
with the polynucleotide of the invention, the gene of the invention
or the vector of the invention.
[0068] The cells obtainable by the method of the invention can be
used, for example, to test drugs according to the methods described
in D. L. Spector, R. D. Goldman, L. A. Leinwand, Cells, a Lab
manual, CSH Press 1998. Furthermore, the cells can be used to study
known drugs and unknown derivatives thereof for their ability to
complement the deficiency caused by mutations in the MRP-1 gene.
For these embodiments the host cells preferably lack a wild type
allele, preferably both alleles of the MRP-1 gene and/or have at
least one mutated from thereof. Ideally, the gene comprising an
allele as comprised by the polynucleotides of the invention could
be introduced into the wild type locus by homologous replacement.
Alternatively, strong overexpression of a mutated allele over the
normal allele and comparison with a recombinant cell line
overexpressing the normal allele at a similar level may be used as
a screening and analysis system. The cells obtainable by the
above-described method may also be used for the screening methods
referred to herein below.
[0069] Furthermore, the invention relates to a polypeptide or
fragment thereof encoded by the polynucleotide of the invention,
the gene of the invention or obtainable by the method described
above or from cells produced by the method described above.
[0070] In this context it is also understood that the variant
polypeptide of the invention can be further modified by
conventional methods known in the art. By providing said variant
proteins according to the present invention it is also possible to
determine the portions relevant for their biological activity or
inhibition of the same. The terms "polypeptide" and "protein" as
used herein are exchangeable. Moreover, what is comprised by said
terms is standard textbook knowledge.
[0071] The present invention furthermore relates to an antibody
which binds specifically to the polypeptide of the invention.
[0072] Advantageously, the antibody specifically recognizes or
binds an epitope containing one or more amino acid substitution(s)
as defined above. Antibodies against the variant polypeptides of
the invention can be prepared by well known methods using a
purified protein according to the invention or a (synthetic)
fragment derived therefrom as an antigen. Monoclonal antibodies can
be prepared, for example, by the techniques as originally described
in Kohler and Milstein, Nature 256 (1975), 495, and Galfr, Meth.
Enzymol. 73 (1981), 3, which comprise the fusion of mouse myeloma
cells to spleen cells derived from immunized mammals. In a
preferred embodiment of the invention, said antibody is a
monoclonal antibody, a polyclonal antibody, a single chain
antibody, human or humanized antibody, primatized, chimerized or
fragment thereof that specifically binds said peptide or
polypeptide also including bispecific antibody, synthetic antibody,
antibody fragment, such as Fab, Fv or scFv fragments etc., or a
chemically modified derivative of any of these. Furthermore,
antibodies or fragments thereof to the aforementioned polypeptides
can be obtained by using methods which are described, e.g., in
Harlow and Lane "Antibodies, A Laboratory Manual", CSH Press, Cold
Spring Harbor, 1988. These antibodies can be used, for example, for
the immunoprecipitation and immunolocalization of the variant
polypeptides of the invention as well as for the monitoring of the
presence of said variant polypeptides, for example, in recombinant
organisms, and for the identification of compounds interacting with
the proteins according to the invention. For example, surface
plasmon resonance as employed in the BIAcore system can be used to
increase the efficiency of phage antibodies which bind to an
epitope of the protein of the invention (Schier, Human Antibodies
Hybridomas 7 (1996), 97-105; Malmborg, J. Immunol. Methods 183
(1995), 7-13).
[0073] In a preferred embodiment the antibody of the present
invention specifically recognizes an epitope containing one or more
amino acid substitution(s) resulting from a nucleotide exchange as
defined supra.
[0074] Antibodies which specifically recognize modified amino acids
such as phospho-Tyrosine residues are well known in the art.
Similarly, in accordance with the present invention antibodies
which specifically recognize even a single amino acid exchange in
an epitope may be generated by the well known methods described
supra.
[0075] In light of the foregoing, in a more preferred embodiment
the antibody of the present invention is monoclonal or
polyclonal.
[0076] The invention also relates to a transgenic non-human animal
comprising at least one polynucleotide of the invention, the gene
of the invention or the vector of the invention as described
supra.
[0077] The present invention also encompasses a method for the
production of a transgenic non-human animal comprising introduction
of a polynucleotide or vector of the invention into a germ cell, an
embryonic cell, stem cell or an egg or a cell derived therefrom.
The non-human animal can be used in accordance with the method of
the invention described below and may be a non-transgenic healthy
animal, or may have a disease or disorder, preferably a disease
caused by at least one mutation in the gene of the invention. Such
transgenic animals are well suited for, e.g., pharmacological
studies of drugs in connection with variant forms of the above
described variant polypeptides since these polypeptides or at least
their functional domains are conserved between species in higher
eukaryotes, particularly in mammals. Production of transgenic
embryos and screening of those can be performed, e.g., as described
by A. L. Joyner Ed., Gene Targeting, A Practical Approach (1993),
Oxford University Press. The DNA of the embryos can be analyzed
using, e.g., Southern blots with an appropriate probe or based on
PCR techniques.
[0078] A transgenic non-human animal in accordance with the
invention may be a transgenic mouse, rat, hamster, dog, monkey,
rabbit, pig, frog, nematode such as Caenorhabditis elegans,
fruitfly such as Drosophila melanogaster or fish such as torpedo
fish or zebrafish comprising a polynucleotide or vector of the
invention or obtained by the method described above, preferably
wherein said polynucleotide or vector is stably integrated into the
genome of said non-human animal, preferably such that the presence
of said polynucleotide or vector leads to the expression of the
variant polypeptide of the invention. It may comprise one or
several copies of the same or different polynucleotides or genes of
the invention. This animal has numerous utilities, including as a
research model for cardiovascular research and therefore, presents
a novel and valuable animal in the development of therapies,
treatment, etc. for diseases caused by cardiovascular diseases.
Accordingly, in this instance, the mammal is preferably a
laboratory animal such as a mouse or rat.
[0079] Thus, in a preferred embodiment the transgenic non-human
animal of the invention is a mouse, a rat or a zebrafish.
[0080] Numerous reports revealed that said animals are particularly
well suited as model organisms for the investigation of the drug
metabolism and its deficiencies or cancer. Advantageously,
transgenic animals can be easily created using said model
organisms, due to the availability of various suitable techniques
well known in the art.
[0081] The invention also relates to a solid support comprising one
or a plurality of the polynucleotide, the gene, the vector, the
polypeptide, the antibody or the host cell of the invention in
immobilized form.
[0082] The term "solid support" as used herein refers to a flexible
or non-flexible support that is suitable for carrying said
immobilized targets. Said solid support may be homogenous or
inhomogeneous. For example, said solid support may consist of
different materials having the same or different properties with
respect to flexibility and immobilization, for instance, or said
solid support may consist of one material exhibiting a plurality of
properties also comprising flexibility and immobilization
properties. Said solid support may comprise glass-, polypropylene-
or silicon-chips, membranes oligonucleotide-conjugated beads or
bead arrays.
[0083] The term "immobilized" means that the molecular species of
interest is fixed to a solid support, preferably covalently linked
thereto. This covalent linkage can be achieved by different means
depending on the molecular nature of the molecular species.
Moreover, the molecular species may be also fixed on the solid
support by electrostatic forces, hydrophobic or hydrophilic
interactions or Van-der-Waals forces. The above described
physico-chemical interactions typically occur in interactions
between molecules. For example, biotinylated polypeptides may be
fixed on a avidin-coated solid support due to interactions of the
above described types. Further, polypeptides such as antibodies,
may be fixed on an antibody coated solid support. Moreover, the
immobilization is dependent on the chemical properties of the solid
support. For example, the nucleic acid molecules can be immobilized
on a membrane by standard techniques such as UV-crosslinking or
heat.
[0084] In a preferred embodiment of the invention said solid
support is a membrane, a glass- or poylpropylene- or silicon-chip,
are membranes oligonucleotide-conjugated beads or a bead array,
which is assembled on an optical filter substrate.
[0085] Moreover, the present invention relates to an in vitro
method for identifying a polymorphism said method comprising the
steps of:
[0086] (a) isolating a polynucleotide or the gene of the invention
from a plurality of subgroups of individuals, wherein one subgroup
has no prevalence for a MRP-1 associated disease and at least one
or more further subgroup(s) do have prevalence for a MRP-1
associated disease; and
[0087] (b) identifying a polymorphism by comparing the nucleic acid
sequence of said polynucleotide or said gene of said one subgroup
having no prevalence for a MRP-1 associated disease with said at
least one or more further subgroup(s) having a prevalence for a
MRP-1 associated disease.
[0088] The term "prevalence" as used herein means that individuals
are be susceptible for one or more disease(s) which are associated
with MRP-1 dysfuntion or dysregulation or could already have one or
more of said disease(s). Thereby, one MRP-1 associated disease can
be used to determine the susceptibility for another MRP-1
associated disease, e.g. altered drug transport may be indicative
for a prevalence for, e.g. cancer. Moreover, symptoms which are
indicative for a prevalence for developing said diseases are very
well known in the art and have been sufficiently described in
standard textbooks such as Pschyrembel.
[0089] Advantageously, polymorphisms according to the present
invention which are associated with MRP-1 dysfunction or
dysregulation or one or more disease(s) based thereon should be
enriched in subgroups of individuals which have a prevalence for
said diseases versus subgroups which have no prevalence for said
diseases. Thus, the above described method allows the rapid and
reliable detection of polymorphism which are indicative for one or
more MRP-1 associated disease(s) or a susceptibility therefor.
Advantageously, due to the phenotypic preselection a large number
of individuals having no prevalence might be screened for
polymorphisms in general. Thereby, a reference sequences comprising
polymorphisms which do not correlate to one or more MRP-1
associated disease(s) can be obtained. Based on said reference
sequences it is possible to efficiently and reliably determine the
relevant polymorphisms.
[0090] In a further embodiment the present invention relates to a
method for identifying and obtaining a pro-drug or a drug capable
of modulating the activity of a molecular variant of a MRP-1
polypeptide comprising the steps of:
[0091] (a) contacting the polypeptide, the solid support of the
invention, a cell expressing a molecular variant gene comprising a
polynucleotide of the invention, the gene or the vector of the
invention in the presence of components capable of providing a
detectable signal in response to drug activity with a compound to
be screened for pro-drug or drug activity; and
[0092] (b) detecting the presence or absence of a signal or
increase or decrease of a signal generated from the pro-drug or the
drug activity, wherein the absence, presence, increase or decrease
of the signal is indicative for a putative pro-drug or drug.
[0093] The term "compound" in a method of the invention includes a
single substance or a plurality of substances which may or may not
be identical.
[0094] Said compound(s) may be chemically synthesized or produced
via microbial fermentation but can also be comprised in, for
example, samples, e.g., cell extracts from, e.g., plants, animals
or microorganisms. Furthermore, said compounds may be known in the
art but hitherto not known to be useful as an inhibitor,
respectively. The plurality of compounds may be, e.g., added to the
culture medium or injected into a cell or non-human animal of the
invention.
[0095] If a sample containing (a) compound(s) is identified in the
method of the invention, then it is either possible to isolate the
compound from the original sample identified as containing the
compound, in question or one can further subdivide the original
sample, for example, if it consists of a plurality of different
compounds, so as to reduce the number of different substances per
sample and repeat the method with the subdivisions of the original
sample. It can then be determined whether said sample or compound
displays the desired properties, for example, by the methods
described herein or in the literature (Spector et al., Cells
manual; see supra). Depending on the complexity of the samples, the
steps described above can be performed several times, preferably
until the sample identified according to the method of the
invention only comprises a limited number of or only one
substance(s). Preferably said sample comprises substances of
similar chemical and/or physical properties, and most preferably
said substances are identical. The methods of the present invention
can be easily performed and designed by the person skilled in the
art, for example in accordance with other cell based assays
described in the prior art or by using and modifying the methods as
described herein. Furthermore, the person skilled in the art will
readily recognize which further compounds may be used in order to
perform the methods of the invention, for example, enzymes, if
necessary, that convert a certain compound into a precursor. Such
adaptation of the method of the invention is well within the skill
of the person skilled in the art and can be performed without undue
experimentation.
[0096] Compounds which can be used in accordance with the present
invention include peptides, proteins, nucleic acids, antibodies,
small organic compounds, ligands, peptidomimetics, PNAs and the
like. Said compounds may act as agonists or antagonists of the
inveniton. Said compounds can also be functional derivatives or
analogues of known drugs. Methods for the preparation of chemical
derivatives and analogues are well known to those skilled in the
art and are described in, for example, Beilstein, Handbook of
Organic Chemistry, Springer edition New York Inc., 175 Fifth
Avenue, New York, N.Y. 10010 U.S.A. and Organic Synthesis, Wiley,
N.Y., USA. Furthermore, said derivatives and analogues can be
tested for their effects according to methods known in the art or
as described. Furthermore, peptide mimetics and/or computer aided
design of appropriate drug derivatives and analogues can be used,
for example, according to the methods described below. Such analogs
comprise molecules may have as the basis structure of known MRP-1
substrates and/or inhibitors and/or modulators; see infra.
[0097] Appropriate computer programs can be used for the
identification of interactive sites of a putative inhibitor and the
polypeptides of the invention by computer assistant searches for
complementary structural motifs (Fassina, Immunomethods 5 (1994),
114-120). Further appropriate computer systems for the computer
aided design of protein and peptides are described in the prior
art, for example, in Berry, Biochem; Soc. Trans. 22 (1994),
1033-1036; Wodak, Ann. N. Y. Acad. Sci. 501 (1987), 1-13; Pabo,
Biochemistry 25 (1986), 5987-5991. The results obtained from the
above-described computer analysis can be used in combination with
the method of the invention for, e.g., optimizing known inhibitors,
analogs, antagonists or agonists. Appropriate peptidomimetics and
other inhibitors can also be identified by the synthesis of
peptidomimetic combinatorial libraries through successive chemical
modification and testing the resulting compounds, e.g., according
to the methods described herein. Methods for the generation and use
of peptidomimetic combinatorial libraries are described in the
prior art, for example in Ostresh, Methods in Enzymology 267
(1996), 220-234 and Dorner, Bioorg. Med. Chem. 4 (1996), 709-715.
Furthermore, the three-dimensional and/or crystallographic
structure of said compounds and the polypeptides of the invention
can be used for the design of peptidomimetic drugs (Rose,
Biochemistry 35 (1996), 12933-12944; Rutenber, Bioorg. Med. Chem. 4
(1996), 1545-1558). It is very well known how to obtain said
compounds, e.g. by chemical or biochemical standard techniques.
Thus, also comprised by the method of the invention are means of
making or producing said compounds. In summary, the present
invention provides methods for identifying and obtaining compounds
which can be used in specific doses for the treatment of specific
forms of MRP-1 associated diseases, e.g. dysfunctions or
dysregulations of the drug transport such as cancer or multidrug
resistance.
[0098] The above definitions apply mutatis mutandis to all of the
methods described in the following.
[0099] In a further embodiment the present invention relates to a
method for identifying and obtaining an inhibitor of the activity
of a molecular variant of a MRP-1 polypeptide comprising the steps
of:
[0100] (a) contacting the protein, the solid support of the
invention or a cell expressing a molecular variant gene comprising
a polynucleotide or the gene or the vector of the invention in the
presence of components capable of providing a detectable signal in
response to drug activity with a compound to be screened for
inhibiting activity; and
[0101] (b) detecting the presence or absence of a signal or
increase or decrease of a signal generated from the inhibiting
activity, wherein the absence or decrease of the signal is
indicative for a putative inhibitor.
[0102] In a preferred embodiment of the method of the invention
said cell is a cell, obtained by the method of the invention or can
be obtained from the transgenic non-human animal as described
supra.
[0103] In a still further embodiment the present invention relates
to a method of identifying and obtaining a pro-drug or drug capable
of modulating the activity of a molecular variant of a MRP-1
polypeptide comprising the steps of:
[0104] (a) contacting the host cell, the cell obtained by the
method of the invention, the polypeptide or the solid support of
the invention with the first molecule known to be bound by a MRP-1
polypeptide to form a first complex of said polypeptide and said
first molecule;
[0105] (b) contacting said first complex with a compound to be
screened, and
[0106] (c) measuring whether said compound displaces said first
molecule from said first complex.
[0107] Advantageously, in said method said measuring step comprises
measuring the formation of a second complex of said protein and
said inhibitor candidate. Preferably, said measuring step comprises
measuring the amount of said first molecule that is not bound to
said protein.
[0108] In a particularly preferred embodiment of the
above-described method of said first molecule is a agonist or
antagonist or a substrate and/or a inhibitor and/or a modulator of
the polypeptide of the invention, e.g., with a radioactive or
fluorescent label.
[0109] In a still another embodiment the present invention relates
to a method of identifying and obtaining an inhibitor capable of
modulating the activity of a molecular variant of a MRP-1
polypeptide comprising the steps of:
[0110] (a) contacting the host cell or the cell obtained by the
method of the invention, the protein or the solid support of the
invention with the first molecule known to be bound by the MRP-1
polypeptide to form a first complex of said protein and said first
molecule;
[0111] (b) contacting said first complex with a compound to be
screened, and
[0112] (c) measuring whether said compound displaces said first
molecule from said first complex.
[0113] In a preferred embodiment of the method of the invention
said measuring step comprises measuring the formation of a second
complex of said protein and said compound.
[0114] In another preferred embodiment of the method of the
invention said measuring step comprises measuring the amount of
said first molecule that is not bound to said protein.
[0115] In a more preferred embodiment of the method of the
invention said first molecule is labeled.
[0116] The invention furthermore relates to a method for the
production of a pharmaceutical composition comprising the steps of
the method as described supra; and the further step of formulating
the compound identified and obtained or a derivative thereof in a
pharmaceutically acceptable form.
[0117] The therapeutically useful compounds identified according to
the methods of the invention can be formulated and administered to
a patient as discussed above. For uses and therapeutic doses
determined to be appropriate by one skilled in the art and for
definitions of the term "pharmaceutical composition" see infra.
[0118] Furthermore, the present invention encompasses a method for
the preparation of a pharmaceutical composition comprising the
steps of the above-described methods; and formulating a drug or
pro-drug in the form suitable for therapeutic application and
preventing or ameliorating the disorder of the subject diagnosed in
the method of the invention.
[0119] Drugs or pro-drugs after their in vivo administration are
metabolized in order to be eliminated either by excretion or by
metabolism to one or more active or inactive metabolites (Meyer, J.
Pharmacokinet. Biopharm. 24 (1996), 449-459). Thus, rather than
using the actual compound or inhibitor identified and obtained in
accordance with the methods of the present invention a
corresponding formulation as a pro-drug can be used which is
converted into its active in the patient. Precautionary measures
that may be taken for the application of pro-drugs and drugs are
described in the literature; see, for review, Ozama, J. Toxicol.
Sci. 21 (1996), 323-329).
[0120] In a preferred embodiment of the method of the present
invention said drug or prodrug is a derivative of a medicament as
defined hereinafter.
[0121] The present invention also relates to a method of diagnosing
a disorder related to the presence of a molecular variant of the
MRP-1 gene or susceptibility to such a disorder comprising
determining the presence of a polynucleotide or the gene of the
invention in a sample from a subject.
[0122] In accordance with this embodiment of the present invention,
the method of testing the status of a disorder or susceptibility to
such a disorder can be effected by using a polynucleotide gene or
nucleic acid of the invention, e.g., in the form of a Southern or
Northern blot or in situ analysis. Said nucleic acid sequence may
hybridize to a coding region of either of the genes or to a
non-coding region, e.g. intron. In the case that a complementary
sequence is employed in the method of the invention, said nucleic
acid molecule can again be used in Northern blots. Additionally,
said testing can be done in conjunction with an actual blocking,
e.g., of the transcription of the gene and thus is expected to have
therapeutic relevance. Furthermore, a primer or oligonucleotide can
also be used for hybridizing to one of the above mentioned MRP-1
gene or corresponding mRNAs. The nucleic acids used for
hybridization can, of course, be conveniently labeled by
incorporating or attaching, e.g., a radioactive or other marker.
Such markers are well known in the art. The labeling of said
nucleic acid molecules can be effected by conventional methods.
[0123] Additionally, the presence or expression of variant MRP-1
gene can be monitored by using a primer pair that specifically
hybridizes to either of the corresponding nucleic acid sequences
and by carrying out a PCR reaction according to standard
procedures. Specific hybridization of the above mentioned probes or
primers preferably occurs at stringent hybridization conditions.
The term "stringent hybridization conditions" is well known in the
art; see, for example, Sambrook et al., "Molecular Cloning, A
Laboratory Manual" second ed., CSH Press, Cold Spring Harbor, 1989;
"Nucleic Acid Hybridisation, A Practical Approach", Hames and
Higgins eds., IRL Press, Oxford, 1985. Furthermore, the mRNA, cRNA,
cDNA or genomic DNA obtained from the subject may be sequenced to
identify mutations which may be characteristic fingerprints of
mutations in the polynucleotide or the gene of the invention. The
present invention further comprises methods wherein such a
fingerprint may be generated by RFLPs of DNA or RNA obtained from
the subject, optionally the DNA or RNA may be amplified prior to
analysis, the methods of which are well known in the art. RNA
fingerprints may be performed by, for example, digesting an RNA
sample obtained from the subject with a suitable RNA-Enzyme, for
example RNase T.sub.1, RNase T.sub.2 or the like or a ribozyme and,
for example, electrophoretically separating and detecting the RNA
fragments as described above. Further modifications of the
above-mentioned embodiment of the invention can be easily devised
by the person skilled in the art, without any undue experimentation
from this disclosure; see, e.g., the examples. An additional
embodiment of the present invention relates to a method wherein
said determination is effected by employing an antibody of the
invention or fragment thereof. The antibody used in the method of
the invention may be labeled with detectable tags such as a
histidine flags or a biotin molecule.
[0124] The invention relates to a method of diagnosing a disorder
related to the presence of a molecular variant of a MRP-1 gene or
susceptibility to such a disorder comprising determining the
presence of a polypeptide or the antibody of the invention in a
sample from a subject.
[0125] In a preferred embodiment of the above described method said
disorder is a cancer disease or a disease related to multidrug
resistance.
[0126] In a preferred embodiment of the present invention, the
above described method is comprising PCR, ligase chain reaction,
restriction digestion, direct sequencing, nucleic acid
amplification techniques, hybridization techniques or immunoassays.
Said techniques are very well known in the art.
[0127] Moreover, the invention relates to a method of detection of
the polynucleotide or the gene of the invention in a sample
comprising the steps of
[0128] (a) contacting the solid support described supra with the
sample under conditions allowing interaction of the polynucleotide
or the gene of the invention with the immobilized targets on a
solid support and;
[0129] (b) determining the binding of said polynucleotide or said
gene to said immobilized targets on a solid support.
[0130] The invention also relates to an in vitro method for
diagnosing a disease comprising the steps of the method described
supra, wherein binding of said polynucleotide or gene to said
immobilized targets on said solid support is indicative for the
presence or the absence of said disease or a prevalence for said
disease.
[0131] The invention furthermore relates to a diagnostic
composition comprising the polynucleotide, the gene, the vector,
the polypeptide or the antibody of the invention.
[0132] In addition, the invention relates to a pharmaceutical
composition comprising the polynucleotide, the gene, the vector,
the polypeptide or the antibody of the invention. These
pharmaceutical compositions comprising, e.g., the antibody may
conveniently be administered by any of the routes conventionally
used for drug administration, for instance, orally, topically,
parenterally or by inhalation. Acceptable salts comprise acetate,
methylester, HCl, sulfate, chloride and the like. The compounds may
be administered in conventional dosage forms prepared by combining
the drugs with standard pharmaceutical carriers according to
conventional procedures. These procedures may involve mixing,
granulating and compressing or dissolving the ingredients as
appropriate to the desired preparation. It will be appreciated that
the form and character of the pharmaceutically acceptable character
or diluent is dictated by the amount of active ingredient with
which it is to be combined, the route of administration and other
well-known variables. The carrier(s) must be "acceptable" in the
sense of being compatible with the other ingredients of the
formulation and not deleterious to the recipient thereof. The
pharmaceutical carrier employed may be, for example, either a solid
or liquid. Exemplary of solid carriers are lactose, terra alba,
sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate,
stearic acid and the like. Exemplary of liquid carriers are
phosphate buffered saline solution, syrup, oil such as peanut oil
and olive oil, water, emulsions, various types of wetting agents,
sterile solutions and the like. Similarly, the carrier or diluent
may include time delay material well known to the art, such as
glyceryl mono-stearate or glyceryl distearate alone or with a
wax.
[0133] The dosage regimen will be determined by the attending
physician and other clinical factors; preferably in accordance with
any one of the above described methods. As is well known in the
medical arts, dosages for any one patient depends upon many
factors, including the patient's size, body surface area, age, the
particular compound to be administered, sex, time and route of
administration, general health, and other drugs being administered
concurrently. Progress can be monitored by periodic assessment.
[0134] Furthermore, the use of pharmaceutical compositions which
comprise antisense-oligonucleotides which specifically hybridize to
RNA encoding mutated versions of the polynucleotitde or gene
according to the invention or which comprise antibodies
specifically recognizing a mutated polypeptide of the invention but
not or not substantially the functional wild-type form is
conceivable in cases in which the concentration of the mutated form
in the cells should be reduced.
[0135] Thanks to the present invention the particular drug
selection, dosage regimen and corresponding patients to be treated
can be determined in accordance with the present invention. The
dosing recommendations will be indicated in product labeling by
allowing the prescriber to anticipate dose adjustments depending on
the considered patient group, with information that avoids
prescribing the wrong drug to the wrong patients at the wrong
dose.
[0136] In another embodiment the present invention relates to the
use of the polynucleotide, the gene, the vector, the polypeptide
the polynucleotides having at a position corresponding to position
926 of the MRP-1 gene (Accession No: U07050) a T insertion, at a
position corresponding to position 79 of the MRP-1 gene (Accession
No: AF022830) an A or at a position corresponding to position
137647 of the MRP-1 gene (Accession No: AC026452) a T, or at a
position corresponding to position 150727 of the MRP-1 gene
(Accession No: AC025277) an A, or the antibody of the invention for
the preparation of a diagnostic composition for diagnosing a
disease.
[0137] A gene encoding a functional and expressible polypeptide of
the invention can be introduced into the cells which in turn
produce the protein of interest. Gene therapy, which is based on
introducing therapeutic genes into cells by ex-vivo or in-vivo
techniques is one of the most important applications of gene
transfer. Suitable vectors and methods for in-vitro or in-vivo gene
therapy are described in the literature and are known to the person
skilled in the art; see, e.g., Giordano, Nature Medicine 2 (1996),
534-539; Schaper, Circ. Res. 79 (1996), 911-919; Anderson, Science
256 (1992), 808-813; Isner, Lancet 348 (1996), 370-374; Muhlhauser,
Circ. Res. 77 (1995), 1077-1086; Wang, Nature Medicine 2 (1996),
714-716; WO94/29469; WO 97/00957 or Schaper, Current Opinion in
Biotechnology 7 (1996), 635-640, and references cited therein. The
gene may be designed for direct introduction or for introduction
via liposomes, or viral vectors (e.g. adenoviral, retroviral) into
the cell. Preferably, said cell is a germ line cell, embryonic
cell, or egg cell or derived therefrom, most preferably said cell
is a stem cell.
[0138] As is evident from the above, it is preferred that in the
use of the invention the nucleic acid sequence is operatively
linked to regulatory elements allowing for the expression and/or
targeting of the polypeptides of the invention to specific cells.
Suitable gene delivery systems that can be employed in accordance
with the invention may include liposomes, receptor-mediated
delivery systems, naked DNA, and viral vectors such as herpes
viruses, retroviruses, adenoviruses, and adeno-associated viruses,
among others. Delivery of nucleic acids to a specific site in the
body for gene therapy may also be accomplished using a biolistic
delivery system, such as that described by Williams (Proc. Natl.
Acad. Sci. USA 88 (1991), 2726-2729). Standard methods for
transfecting cells with recombinant DNA are well known to those
skilled in the art of molecular biology, see, e.g., WO 94/29469;
see also supra. Gene therapy may be carried out by directly
administering the recombinant DNA molecule or vector of the
invention to a patient or by transfecting cells with the
polynucleotide or vector of the invention ex vivo and infusing the
transfected cells into the patient.
[0139] In a further embodiment the present invention relates to the
use of the polynucleotide, the gene, the vector, the polypeptide
the polynucleotides having at a position corresponding to position
926 of the MRP-1 gene (Accession No: U07050) a T insertion, at a
position corresponding to position 79 of the MRP-1 gene (Accession
No: AF022830) an A or at a position corresponding to position
137647 of the MRP-1 gene (Accession No: AC026452) a T, or at a
position corresponding to position 150727 of the MRP-1 gene
(Accession No: AC025277) an A, or the antibody of the invention for
the preparation of a pharmaceutical composition for treating a
disease.
[0140] In a more preferred embodiment of the use of the present
invention said disease is cancer or a disease related to multidrug
resistance.
[0141] Finally, the present invention relates to a diagnostic kit
for detection of a single nucleotide polymorphism comprising the
polynucleotide, the gene, the vector, the polypeptide, the
antibody, the host cell, the transgenic non-human animal or the
solid support of the invention.
[0142] The kit of the invention may contain further ingredients
such as selection markers and components for selective media
suitable for the generation of transgenic cells and animals. The
kit of the invention can be used for carrying out a method of the
invention and could be, inter alia, employed in a variety of
applications, e.g., in the diagnostic field or as research tool.
The parts of the kit of the invention can be packaged individually
in vials or other appropriate means depending on the respective
ingredient or in combination in suitable containers or
multicontainer units. Manufacture of the kit follows preferably
standard procedures which are known to the person skilled in the
art. The kit may be used for methods for detecting expression of a
mutant form of the polypeptides, genes or polynucleotides in
accordance with any one of the above-described methods of the
invention, employing, for example, immunoassay techniques such as
radioimmunoassay or enzymeimmunoassay or preferably nucleic acid
hybridization and/or amplification techniques such as those
described herein before and in the Examples as well as
pharmacokinetic studies when using non-human transgenic animals of
the invention.
[0143] The figures illustrate the invention:
[0144] FIG. 1: The figure shows, where the novel MRP-1 SNP's are
located on the gene and the protein, respectively.
[0145] FIG. 2: The figure illustrates the correlation between MRP-1
transport activity, intracellular carcinogen/drug concentrations
and cancer risk, therapy outcome and side effects.
[0146] FIG. 3: Diagram 1A and 1B represent the correlation of the
genotype (wt/wt: 1; wt/mut and mut/mut:2) with MRP-1 mRNA content
in duodenal biopsies from healthy volunteers derived from two
independent experiments, before (A) and after (B) application of
rifampicin. The p-value of the statistical evaluation
(Kruskal-Wallis-Test), which result in a genotyp/phenotype
correlation is p=0.086. The p-value of the paired T-test
(p<0.001) demonstrates, that rifampicin has no effect on MRP-1
mRNA expression. Thus, the differences in the MRP-1 mRNA content
are based on interindividual differences. The statistical analyses
were performed using the computer program SPSS 10.0 (SPSS, Chicago,
USA).
[0147] The invention will now be described by reference to the
following biological Examples which are merely illustrative and are
not constructed as a limitation of the scope of the present
invention.
EXAMPLE 1
Isolation of Genomic DNA from Human Blood, Generation and
Purification of MRP-1 Fragments
[0148] Genomic DNA was obtained by standard ion exchange
chromatography techniques (Quiagen kits for isolation of genomic
DNA from blood). Specific oligonucleotide primers, 2 for each
fragment, were applied to obtain defined DNA fragments by
polymerase chain reaction (PCR) containing specific parts of the
MRP-1 gene. These specific oligonucleotide primers were designed to
bind to sequences upstream and downstream of various exons of the
gene. The resulting DNA fragments were to encode not only exon
sequences, but also some intron sequences at the exon-intron
boundaries. Such intronic sequences adjacent to the exons are known
to be important for correct splicing and subsequent expression of
the mRNA, which encodes for the respective protein. Oligonucleotide
primer pairs that were optimized for each of the PCR fragments,
synthesized and purified by affinity chromatography (OPC
cartridges). The primer sequences for the amplification of the
single fragments are listed in Table 1.
[0149] Polymerase chain reactions for the single MRP-1 gene
fragments, were performed under conditions, that were optimized for
each of these fragments. These MRP-1 gene fragments cover the
respective exons, as well as regulatory regions, like promoter,
5'-UTR and 3'-UTR (see Table 1). PCRs were carried out for all
fragments in a reaction volume of 50 .mu.l. 40 ng DNA template was
added to standard PCR buffer containing 1.5 mM MgCl2 (Qiagen,
Hilden), 200 .mu.M dNTP's (Roth, Karlsruhe), 0.4 .mu.M (conditions
A and C) or 1.6 .mu.M (condition B) of each primer (Metabion,
Munich), 10 .mu.l Q-Solution (condition C; Qiagen, Hilden), 4 .mu.l
DMSO (condition B) and 1 U Taq polymerase (Qiagen, Hilden). All
PCRs (conditions A and C) were performed on a Perkin Elmer
thermocycler (model 9700) with an initial denaturation step of 2
min at 94.degree. C. and 34 amplification cycles of denaturation at
94.degree. C. for 45 sec, primer annealing at 62.degree. C. for 45
sec, and 1 min for 72.degree. C. followed by a final extension of
72.degree. C. for 10 min. In the case of condition B the PCR
reaction was performed with an initial denaturation step of 3 min
at 96.degree. C. and 35 amplification cycles of denaturation at
96.degree. C. for 45 sec, primer annealing at 62.degree. C. for 30
sec, and 1 min for 72.degree. C. followed by a final extension of
72.degree. C. for 10 min.
[0150] The optimized PCR-conditions and the resulting size of the
desired and obtained fragments are listed in Table 1. The defined
DNA fragments containing specific parts of the human MRP-1 gene
were processed to remove nonincorporated nucleotides and buffer
components that otherwise interfere with the subsequent
determination of the individual MRP-1 genotype by direct DNA
sequencing. For this purification, standard ion exchange
chromatography techniques were used (Quiagen kits for PCR fragment
purification). For all of the fragments, sufficient yields of
purified fragments, suitable for direct DNA sequence analyses, were
obtained.
EXAMPLE 2
Identification of Different MRP-1 Gene Alleles by Sequence
Determination in Various Individuals
[0151] For sequence analysis of relevant regions of the human MRP-1
gene from 24 different individuals, PCR amplification of the
relevant fragments of this gene was carried out (see Table 1) and
the purified PCR products subsequently sequenced with established
methods (ABI dyeterminator cycle sequencing). A very important
parameter that was needed to consider using this approach was that
each normal human individual harbors two copies of this gene.
Because of this diploidy (of autosomal genes; the MRP-1 gene is an
autosomal gene on chromosome 16), great care had to be taken in the
evaluation of the sequences to be able to identify unambiguously
not only homozygous sequence variations but also heterozygous
variations.
[0152] For the initial evaluation of gene variations in the human
population, sequence analyses of the relevant regions of the MRP-1
gene were carried out from the genomic DNA from 24 different
individuals. This number of individual samples was then extended
for a screening for all the MRP-1 gene fragments, in which SNP's
could be identified. The sequences were inspected for the occurence
of DNA sequences that were deviant from the published sequences of
the MRP-1 gene. These reference sequences are considered as
"wildtype" sequences in all of this work. Because population
genetics enables a calculation of the expected frequency of
homozygous vs. heterozygous alleles of a defined gene
(Hardy-Weinberg distribution, using the formulas
p=(2.times.AA+1.times.Aa- )/2N and p+q=1:AA=number of probands
homozygous for the wt-allele, Aa=number of heterozygotes, N=size of
the sample test, p=frequency of the wt-allele, q=frequency of the
mut-allele, q.sup.2=frequency of the genotype homozygous for the
mut-allele), it was possible to confirm the predicted (using these
formulas) distribution of homozygous vs. heterozygous alleles and
deviations with the experimental findings (see Table 2). This
serves as internal control and confirmation that a detected
sequence deviation indeed represents a novel allele.
[0153] In total 42 new and still unpublished polymorphisms could be
found in the MRP-1 gene. The localisation of these novel SNP's in
the MRP-1 gene and in the MRP-1 protein, respectively, is shown in
FIG. 1. 6 of all these new polymorphisms could be identified only
in renal cell carcinoma (RCC) samples (see also example 4). The
following table gives an overview over all different types of novel
MRP-1 polymorphisms, which have been identified in the initial
screen (24 control samples, example 2), as well as in the extended
screen, that includes clinical samples (70 RCC samples, see example
4):
1 Total number SNP location of newly found SNP's comments Promoter:
11 2 SNP's in RCC samples only Introns: 20 2 SNP's in RCC samples
only Exons: total 10 silent 7 1 SNP in RCC samples only amino acid
3 R723Q (splicing variant substitution region, first ATP binding
domain) R433S (cytoplasmic domain) F329C (transmembrane domain no.
6; in RCC samples only) 3'-UTR 1
[0154] In regard to the 42 newly found SNP's, the different types
of polymorphisms that were detected, as well as their distribution
over the MRP-1 gene and the possible meaning of the new SNP's are
described in more detail below. The exact positions and further
details of the novel alleles, including the exact novel sequence
and sequence deviation, and the homozygous vs. heterozygous
distribution of the respective allele in the population are listed
in Table 2. The expected frequency for homozygotes of the variant
allele were calculated on the basis of the Hardy-Weinberg
distribution (formulas see above). The deviant base in the sequence
is bold and underlined.
[0155] The polymorphisms newly found in the MRP-1 gene might have
an effect either on the function of the MRP-1 polypeptide or its
expression or translation. The promoter polymorphisms may
especially affect the transcription level, while the SNP which was
identified in the 3'-UTR might have an effect on the stability of
the respective mRNA. Because the amino acid substitutions F329C,
R433S and R723Q are localized in specific functional domains of the
MRP-1 polypeptide (see above in the table), an effect of these
SNP's on folding, activity or substrate specificity of the
repective domains is conceivable. The single nucleotide
polymorphisms resulting in silent mutations may effect interaction
with a tRNA during translation of mRNA encoded by a gene comprising
said single nucleotide polymorphisms. The polymorphisms, which
could be found in the introns of the MRP-1 gene might have an
effect on splicing of MRP-1 transcripts containing said single
nucleotide polymorphisms.
[0156] The described single nucleotide polymorphisms are useful as
e.g. diagnostic markers since they could be correlated with
phenotypes resulting thereof, such as cancer, like kidney cancer.
Furthermore the single nucleotide polymorphisms in MRP-1 may cause
unsufficient and/or altered drug uptake, transport or
elimination.
EXAMPLE 3
Methods for Specific Detection and Diagnosis of MRP-1 Alleles
[0157] Methods to detect the various MRP-1 alleles that have been
identified utilize the principle that specific sequence differences
can be translated into reagents for allele differentiation. These
reagents provide the necessary backbone for the development of
diagnostic tests. Examples for such reagents include--but are not
limited to--oligonucleotides that deviate from the wildtype MRP-1
sequence in the newly identified base substitution. Frequently, the
principles of diagnostic tests for the determination of the
individual MRP-1 gene status include--but are not limited
to--differences in the hybridization efficiencies of such reagents
to the various MRP-1 alleles. In addition, differences in efficacy
of such reagents in, or as different substrates for, enzymatic
reactions, e.g. ligases or polymerases or restriction enzymes can
be applied. The principles of these are well known to experts of
the field. Examples are PCR- and LCR techniques,
Chip-hybridizations or MALDI-TOF analyses. Such techniques are
described in the prior art, e.g., PCR technique: Newton, (1994)
PCR, BIOS Scientific Publishers, Oxford; LCR-technique: Shimer,
Ligase chain reaction. Methods Mol. Biol. 46 (1995), 269-278; Chip
hybridization: Ramsay, DNA chips: State-of-the art. Nature
Biotechnology 16 (1998), 40-44; and MALDI-TOF analysis: Ross, High
level multiplex genotyping by MALDI-TOF mass spectrometry, Nature
Biotechnology 16 (1998), 1347-1351. Other test principles are based
on the application of reagents that specifically recognize the
MRP-1 variant as translated expressed protein. Examples are
allele-specific antibodies, peptides, substrate analogs,
inhibitors, or other substances which bind to (and in some
instances may also modify the action of) the various MRP-1 protein
forms that are encoded by the new MRP-1 alleles. The examples that
are presented here, to demonstrate the principles of diagnostic
tests with reagents derived from the novel nucleotide substitutions
defined in this application, are based on PCR-methods. It is
obvious that, applying the described specific reagents, any of the
other methods will also work for the differentiation of MRP-1
alleles.
EXAMPLE 4
Distribution of MRP-1 Single Nucleotide Polymorphisms in Kidney
Cancer Samples
[0158] To identify potential direct correlations of MRP-1
polymorphisms with clinical relevant phenotypes in humans, totally
70 renal cell carcinoma (RCC) samples were subjected to the
determination of MRP-1 polymorphisms as described in example 2.
Kidney cancer is the third most frequent urological tumor,
accounting in the United States for 28.000 cases in the year 1995
and approximately 11.000 deads each year in the US (Wingo et al.
1995, CA Cancer J Clin 45 (1): 8-30). One of the major risk factors
for sporadic RCC are somatic mutations in the VHL tumor suppressor
gene (Levine 1996, Radiol Clin North Am 34: 947-964; Linehan et al.
1995, JAMA 273: 564-570). The incidence of kidney cancer increases
continuously by 2 to 4% per year in the United States and other
industrialized countries (Chow et al. 1999, JAMA 281 (17):
1628-1631). These data support, that environmental factors, i.e.
exposure to carcinogens, diuretic and antihypertensive drugs,
tobacco smoke and dietary constituents may be involved in the
occurence of RCC (Schlehofer et al. 1996, Int. J. Cancer 66:
723-726; Heath et al. 1997, Am. J. Epidemiol 145 (7): 607-613).
[0159] As excretory organs the kidneys are committed to the
detoxification and excretion of carcinogens and metabolites. It is
feasible to assume that factors or genes that play a role in the
defense of kidney cells against dietary and environmental toxins or
metabolites may influence the individual susceptibility towards
RCC. Consequently, genetic polymorphisms in xenobiotic-metabolizing
enzymes have been reported to modify RCC risk in the Caucasian
population (Longuemaux et al. 1999, Cancer Res. 59: 2903-2908). Due
to its role in detoxification, the gene for the human multidrug
resistance-associated protein (MRP-1) may be another interesting
candidate. For the evaluation, if some of the newly found MRP-1
single nucleotide polymorphisms are overrepresented and
underrepresented in these kidney cancer samples, respectively, the
allele distribution was determined. The allele, as well as the
genotype frequencies for all new MRP-1 polymorphisms distributed on
the kidney cancer samples and in comparison to that distributed on
control samples are listed in the following table.
2 Frequency in % Homozygotes Frequency mutant in % (expected Sample
Wt- Mut- Homozygotes Hardy SNP collection allele allele
heterozygotes mutant Weinberg) T124667C Controls 62.5 37.5 50 12.5
14.1 (intron 1) RCC G1884A Controls 93.3 6.7 13.3 0 0.5 (Prom1/exon
1) RCC 1720-1723delGGTA Controls 87.5 12.5 25 0 1.5 (Prom 2) RCC
83.6 16.4 23.4 4.7 2.7 C1163T Controls 91.3 8.7 17.4 0 0.7 (Prom 3)
RCC 84.5 15.5 27.7 1.7 2.4 926insT Controls 82.4 17.6 11.8 11.8 3.1
(Prom 3) RCC 62.9 37.1 51.6 11.3 13.8 437insTCCT Controls 97.6 2.4
4.8 0 0.1 TCC(Prom 4) RCC 96.3 3.7 7.4 0 0.1 A381G Controls 72.7
27.3 36.4 9.1 7.4 (Prom 5) RCC 61.5 38.5 50.8 13.1 14.8 G233A
Controls 84.8 15.2 30.4 0 2.3 (Prom 5) RCC 77.9 22.1 34.4 4.9 4.9
C189A Controls 95.7 4.3 8.7 0 0.2 (Prom 5) RCC G39508A Controls
93.5 6.5 13.04 0 0.4 (intron 2) RCC 92.7 7.3 11.3 1.6 0.5 C174T
Controls 95.8 4.2 8.3 0 0.2 (intron 6) RCC 100 0 0 0 0 C248A
Controls 79.2 20.8 25 8.3 4.3 (intron 7) RCC 80 20 40 0 4 C258G
Controls 70.8 29.2 33.3 12.5 8.5 (intron 7) RCC 71.8 28.2 45.5 5.5
7.9 G79A (exon Controls 93.7 6.3 12.5 0 0.4 8, Pro to Pro) RCC 96.3
3.7 7.5 0 0.1 T88C (exon Controls 72.9 27.1 37.5 8.3 7.3 8, Val to
Val) RCC 71.3 28.7 42.6 7.4 8.2 T249G (exon RCC 99.3 0.7 1.5 0 0.01
8, (only in Phe329Cys) these samples) *T95C (exon Controls 71.7
28.3 39.1 8.7 7.9 9, Asn to Asn) RCC 73.1 26.9 44.8 4.5 7.2 *A259G
Controls 71.7 28.3 39.1 8.7 7.9 (intron 9) RCC 73.9 26.1 43.3 4.5
6.8 G57998T Controls 96.9 3.1 6.3 0 0.1 (exon 10, Arg433Ser) RCC
99.3 0.7 1.5 0 0.01 C57853T Controls 97.9 2.1 4.2 0 0.1 (intron 10)
RCC 97.1 2.9 5.8 0 0.1 C53282G Controls 77.1 22.9 37.5 4.2 5.3
(intron 11) RCC 73.8 26.2 46.2 3.1 6.8 *A137710G Controls 79.2 20.8
33.3 4.2 4.4 (intron 12) RCC 81.5 18.5 29.6 3.7 3.4 *C137667T
Controls 79.2 20.8 33.3 4.2 4.4 (exon 13, Leu to Leu) RCC 81.5 18.5
29.6 3.7 3.4 C137647T Controls 85.4 14.6 29.2 0 2.1 (exon 13, Tyr
to Tyr) RCC 94.4 5.6 7.4 1.9 0.3 *G27258A Controls 95.8 4.2 8.3 0
0.2 (exon 17, Arg723Gln) RCC 96.2 3.8 7.7 0 0.2 *34207delAT
Controls 95.8 4.2 8.3 0 0.2 (intron 18) RCC 96.3 3.7 7.4 0 0.1
G34215C Controls 84.8 15.2 30.4 0 2.3 (intron 18) RCC 84.3 15.7
25.7 2.9 2.5 55156insTG Controls 75 25 0 25 6.3 GGC (intron 21) RCC
77.6 22.4 0 22.4 5.03 T55472C Controls 83.3 16.7 8.3 12.5 2.8
(intron 22) RCC 78.6 21.4 10.7 16.1 4.6 G14008A Controls 80.4 19.6
39.1 0 3.8 (exon 28, Ser to Ser) RCC 73.3 26.7 44.2 4.7 7.2
G150727A Controls 66.7 33.3 50 8.3 10.9 (intron 28) RCC 55 45 44.3
22.9 20.3 17970delT Controls 75 25 41.7 4.2 6.3 (intron 29) RCC
75.7 24.3 34.3 7.2 5.9 G18195A Controls 73.3 26.7 40 6.7 7.1
(intron 30) RCC 80.4 19.6 21.7 8.7 3.8 G21133A (3' Controls 97.9
2.1 4.2 0 0.1 flanking region) RCC 95.7 4.3 8.7 0 0.2 G38646C
Controls 73.3 26.7 53.3 0 7.1 (Prom 1) RCC G34218A RCC 96.3 3.7 7.4
0 0.1 (intron 18) (only in these samples) C18067T RCC 98.9 1.1 2.2
0 0.02 (exon 30, Ala (only in to Ala) these samples) C440T RCC 99.3
0.7 1.5 0 0.01 (Prom 5) (only in these samples) C1625A RCC 96.9 3.1
6.3 0 0.1 (prom 2) (only in these samples) C17900T RCC 97.9 2.1 4.3
0 0.1 (intron 29) (only in these samples)
[0160] Three pairs of linked polymorphisms are listed in this
table, whereas each SNP is marked by an asterics. In regard to
their under- and overrepresentation in the RCC samples in
comparison to the control samples, respectively, all of the new
single nucleotide polymorphisms are of great interest, because they
represent genetic variety in humans, which may serve as potential
targets for diagnosis and therapy and as risk factors for kidney
cancer. Some examples: in contrast to the control samples the
mutant alleles of 4 promoter SNP's found in the MRP-1 gene (C1163T
(Prom 3), 926insT (Prom 3), A381G (Prom 5) and G233A (Prom 5)) are
overrepresented in the RCC sample group. Likewise some of the new
intron SNP's, like G150727A (intron 28) and T55472C (intron 22), as
well as the silent mutation G14008A (exon 28, Ser to Ser) show
allele distributions, which point to correlation with kidney
cancer. In addition, especially the 6 SNP's, which could be only
detected in the RCC samples may have an impact for the diagnosis
and therapy of kidney cancer.
EXAMPLE 5
Statistical Analyses of Correlations between MRP-1 Single
Nucleotide Polymorphisms and Renal Cell Carcinoma (RCC)
[0161] Statistical evaluations were performed in regard to the
presence of SNP's in RCC samples compared to their frequencies in a
control population. For this purpose, 70 RCC samples and 24 control
samples were compared. Statistical analysis was performed using the
computer programm SPSS 10.0 (SPSS, Chicago, USA). This evaluation
results in statistically significant correlations of definite SNP's
with the existence of renal cell carcinoma (RCC).
[0162] The p-values of the statistical evaluation
(Chi-Quadrat-Test), which result in genotype/phenotype correlations
are:
3 gene SNP Controls vs. RCC, p-value MRP-1 926insT (Promoter) 0.005
G79A (exon 8) 0.063 C137647T (exon 0.039 13)
EXAMPLE 6
Effects of Kidney Cancer Associated MRP-1 Polymorphisms on Drug
Transport Activity and Pharmacology
[0163] As excretory organs the kidneys are committed to the
detoxification and excretion of watersoluble carcinogens and
metabolites. Therefore, factors or genes that influence the
individual susceptibility towards kidney cancer are related to the
defense capacity of kidney cells against dietary and environmental
toxins or metabolites (Epidauros MDR-1 risk factor patent). Among
these factors, the gene for the P-glycoprotein (Pgp), which
transports toxic substances, has been shown to confer a significant
risk factor for kidney cancer, such as for RCC, if it is present in
an allelic version that corresponds to low transport activity
(Epidauros MDR-1 risk factor patent).
[0164] The multidrug resistance-associated protein 1 (MRP-1) is,
like MDR-1 expressed in the renal tubular cells of the kidney and
extrude different classes of substances in an ATP dependent manner
from the inside to the outside of plasma membranes within these
cells. The physiological role of this energy-dependent export
mechanism in the kidney is the protection of cells. The fact that,
like MDR-1 SNP's, also polymorphisms in the MRP-1 gene (which has a
very similar function) confer significantly increased risk to
develop kidney cancer, such as RCC (see tables in examples 2, 4 and
the results of the statistical evaluation in example 5,
respectively), indicates the underlying molecular mechanism to be
the same for the functional polymorphisms in MDR-1 as well as in
MRP-1: altered and/or reduced transport capacities lead to
increased exposure of renal cells to carcinogenic, toxic and/or
noxic substances, which is responsible for the increased risk to
develop malignant changes in tubular cells. Beside the Promoter SNP
926insT, which shows a statistically significant correlation with
RCC (p=0.005), also the following MRP-1 promoter SNP's C1163T,
A381G and G233A, which are overrepresented in RCC are good
candidates for such risk factors.
[0165] Variable transport capacities of MRP-1-variants play a role
not only in influencing the individual risk of developing kidney
cancer, such as RCC, but such variations will also affect
individual pharmacological responses to medications. For example,
the expression of MRP-1 correlates with therapy outcome in cancer
therapy: Higher MRP-1 activity leads to a resistance of the cell
against MRP-1 substrates. This multidrug resistance could be shown
for numerous MRP-1 substrates.
[0166] Therefore, MRP-1 polymorphisms, especially those with
functional importance, even up to a degree that associated with
increased risk for kidney cancer, such as RCC due to a decreased
capacity of tubular cells to clear damaging agents, are important
for predicting clearance and uptake of MRP-1 substrates, or drugs
whose metabolites are MRP-1 substrates (see FIG. 2A and B).
EXAMPLE 7
Correlation of MRP-1 Polymorphisms with MRP-1 Expression and Side
Effects during Therapy with MRP-1 Substrates
[0167] Functional polymorphisms in the MRP-1 gene (see tables in
examples 2 and 4) affect the transport activity and subsequently
the levels of drugs which are substrates of MRP-1. Increased levels
of such drugs can lead to side effects whereas decreased levels may
result in subtherapeutical drug levels that lead to therapy
failure. Three different patient collectives, two show side effects
during drug therapy and one for which the MRP-1 mRNA levels had
been defined, were analyzed to determine whether MRP-1
polymorphisms correlate with transporter activity and subsequently
with alterations in drug activities and side effects. Statistical
evaluations were performed in regard to the presence of SNP's in
these collectives with side effects during drug therapy and
increased/decreased mRNA levels compared to their frequencies in
control samples. For this purpose, the 3 collectives (collective 1:
samples with nephrotoxicities after cisplatin therapy; collective
2: liver and kidney side effects; collective 3: samples with
defined high or low MRP-1 mRNA levels) were screened for all MRP-1
gene fragments, in which the new SNP's could be detected. For those
of the newly identified MRP-1 SNP's which are overrepresented or
underrepresented, the allele distribution was determined. As an
example, the allele and genotype frequencies for one MRP-1
polymorphism are listed in the following table for collective 2 and
compared to control samples:
4 Frequency in % Homozygotes Frequency mutant in % (expected Sample
Wt- Mut- Homozygotes Hardy SNP collection allele allele
heterozygotes mutant Weinberg) G150727A Controls 66.7 33.3 50 8.3
10.9 (intron 28) Collective 2 50 50 14.3 42.9 25
[0168] In contrast to control samples the mutant allele (150727A)
of one SNP found in the MRP-1 gene (G150727A, intron 28) is
overrepresented in the samples of collective 2. Statistical
evaluations were performed in regard to the presence of this SNP in
samples with liver and kidney side effects (collective 2) compared
to their frequencies in a control population. The statistical
analysis was performed using the computer program SPSS 10.0 (SPSS,
Chicago, USA). This evaluation results in a statistically
significant correlation of a definite SNP with liver and kidney
side effects.
[0169] The genotyp/phenotype correlation is confirmed by the
p-value of the statistical evaluation (Chi-Quadrat-Test):
5 Controls vs. liver and kidney gene SNP side effects, p-value
MRP-1 G150727A (intron 28) 0.044
[0170] Furthermore, a correlation of MRP-1 gene variants and mRNA
expression of MRP-1 could be found for two new MRP-1 SNP's (T95C,
exon 9, Asn to Asn and A259G, intron 9). These are linked SNP's
(see also table in example 4). As shown in FIG. 3 (Diagramm A and
B), the mutant allele correlates with decreased MRP-1 mRNA
expression. Thus, the analysis of these functional important SNP's
is of high diagnostic/prognostic value, because it allows the
prediction of therapy outcome and side effects, and of expression
levels of MRP-1.
EXAMPLE 8
MRP1 Genotypes in Patients Suffering from Drug-Induced Hepatic
Toxicity
[0171] MRP1 genotypes were investigated in patients suffering from
drug-induced hepatic toxicity (n=7) and healthy controls (n=95).
Pearson chi-square was calculated from contingency tables to test
the equality of proportions between patients and controls. When
appropiate Fisher's Exact Test was applied. The level of
significance was set to p=0.05. Statistical analysis was performed
using SPSS 10.1 (SPSS, Chicago, USA). The level of significance was
set to p=0.05.
[0172] Three SNPs (T>C.sub.95, A>G.sub.259, and
C>G.sub.53282) were found to be associated with the occurance of
liver toxicity. The frequency of homozyguosly mutant genotypes was
statistically significant elevated as summarized in the following
table.
6 Frequency Distribution of MRP1 Genotypes SNP Controls [%]
Controls [%] wt > mut.sub.position AccNo.sup.1 SeqID N wt/wt
wt/m m/m N wt/wt wt/m m/m P.sup.2 T > C.sub.95 AF022831 171 7
47.8 44.6 7.6 92 85.7 14.3 0.035 A > G.sub.259 AF022831 177 7
47.8 44.6 7.6 92 85.7 14.3 0.035 C > G.sub.53282 GI: 7209451 195
6 55.3 40.4 4.3 94 85.3 16.7 0.05 .sup.1Accession Number of
reference sequence (wt allele) .sup.2P value of statistical test
wt/wt homozygous wildtypes wt/m heterozygots m/m homozygous
mutants
[0173] Two of these SNPs are linked (T>C.sub.95 and
A>G.sub.259) and have been demonstrated (example 7) to correlate
with decreased MRP1 expression. It can be concluded that a reduced
hepatic MRP1 expression leads to a decreased capacity of
hepatocytes to transport toxic substrates with the consequence of
an elevated risk to hepatocellular damage. Thus, SNPs in the MRP1
can explain interindividual variations in the susceptibility to
adverse drug events (ADEs) and are important diagnostic markers to
predict the individual risk of patients in order to prevent
patients from ADEs by e.g. dosage adjustments or switching to other
medications.
EXAMPLE 9
MRP1 Genotypes in Patients Suffering from Renal Carcinoma (RCC)
[0174] MRP1 genotypes were investigated in patients suffering from
renal carcinoma (RCC) and healthy controls. Pearson chi-square was
calculated from contingency tables to test the equality of
proportions between RCC and controls. When appropiate Fishers Exact
Test was applied. The level of significance was set to p=0.05.
Statistical analysis was performed using SPSS 10.1 (SPSS, Chicago,
USA). Pearson chi-square was calclated to test the equality of
proportions. The level of significance was set to p=0.05.
[0175] Three SNPs have been already described to be correlated with
RCC in example 5. Additionally, the nucleotide substitution
A>G.sub.381 was found to be statistically significant associated
with RCC and T>C.sub.124667 tended to be associated with renal
carcinoma confirming further the important role of MRP1 for
pharmacology and toxicology of drugs.
7 Frequency Distribution of MRP1 Genotypes SNP Patients [%]
Controls [%] wt > mut.sub.position AccNo.sup.1 SeqID N wt/wt
wt/m m/m N wt/wt wt/m m/m P.sup.2 T > C.sub.124667 AC026452 075
33 45.5 51.5 3.0 90 57.8 31.1 11.1 0.075 A > G.sub.381 U07050
111 59 35.6 52.5 11.9 88 53.6 32.1 14.3 0.027 .sup.1Accession
Number of reference sequence (wt allele) .sup.2P value of
statistical test wt/wt homozygous wildtypes wt/m heterozygots m/m
homozygous mutants
[0176]
8TABLE 1 Primers for the amplification of fragments of the MRP1
gene Primer sequence PCR fragment (5' to 3' PCR Fragment name PCR
primer position orientation) condition size Accession number
AC026452 Exon 1/Prom 1 38590-38608 MRP1-P1f GTA GGG GGC TCC GTT CAC
G B 880 bp 124576-124600 MRP1-E1r2 CCT GGA AGG TTG TTT TTA CAG ACG
G Accession number U07050 Promoter fragment 1359-1377 MRP1-P2f TGG
AGA CTG GCG CCG TCT G C 408 bp 2 1767-1746 MRP1-P2r AAG GAC AGT ATC
CGT CAC CAG G Promoter fragment 830-851 MRP1-P3f CAT GGG GTT GTG
AGG ATT GCA C A 590 bp 3 1423-1401 MRP1-P3r TGA GAT TCA AAC CCG TGA
GCA GC Promoter fragment 351-374 MRP1-P4f CTT AGA AAC TCA TTC ACC
CTT A 550 bp 4 GGG 902-881 MRP1-P4r GTG ACA AGG CTT CCT AAG GCT G
Promoter fragment 144-170 MRP1-P5f GAT TAA CAT CTG CCA TCT TAC CAT
A 321 bp 5 AAG 465-445 MRP1-P5r CCT CCC CCC AAT CAA AGG ACC GI
number 7209451 Exon 2 39769-39789 MRP1-E2f1 AGC TGG TTT CAT GCT CCA
GGC A 374 bp 39416-39440 MRP1-E2r1 CTA GAA GAA GGA ACT TAG GGT CAA
C Accession number AF022825 Exon 3 24-44 MRP1-E3f TTC CAG GGC GGT
CTG TTG TAG A 233 bp 257-235 MRP1-E3r ATT ACT TTT GGT CTC CAC TGA
GC Accession number AF022826 Exon 4 68-90 MRP1-E4f2 AAA ACC CAA CAA
CTC CTG TCT TG A 230 bp 297-278 MRP1-E4r GCA TCT TTC CCT CCG GGT CC
Accession number AF022827 Exon 5 35-55 MRP1-E5f1 ACC CAG CCC CAG
AAT GTG ATC A 206 bp 240-219 MRP1-E5r2 GCA CAC ACA CTC ATT TGT GGT
C Accession number AF022828 Exon 6 4-26 MRP1-E6f GAG CAG CTG ACT
ACT TGC TAA GC A 209 bp 212-190 MRP1-E6r1 CAT TCA TTC ATT CAC TCC
CCA CC Accession number AF022829 Exon 7 17-41 MRP1-E7f CTG TCA TTG
ACT CTC ATT GCC TAA A 279 bp C 295-275 MRP1-E7r1 AGT AAC AGG CAG
CAC TGC CAG Accession number AF022830 Exon 8 29-49 MRP1-E8f ATC TCT
GGC AGA CCC CAC AAC A 336 bp 364-341 MRP1-E8r1 AAC TGA AAG ATC AAA
GCC AAG GAG Accession number AF022831 Exon 9 26-47 MRP1-E9f CCC CAC
GTG TCA CAA GTC ATT C A 322 bp 347-328 MRP1-E9r TGG GCT GGA AAT CCC
CAC GC GI number 7209451 Exon 10 58203-58184 MRP1-E10f1 GGG AGG AGG
AGA GAT CTG CG A 413 bp 57791-57810 MRP1-E10r1 TGA ACC ACA GCC GGA
ACT GC GI number 7209451 Exon 11 53578-53559 MRP1-E11f GGA TGG ATC
AAC CGG GGA AG A 353 bp 53226-53248 MRP1-E11r TCA GAA TCC CAG ATA
TGC AGC CG GI number 7209451 Exon 12 22183-22204 MRP1-E12f1 TGT TGA
GTG ATG GGC TGA TCC C A 344 bp 22526-22499 MRP1-E12r CCT TTT AAA
AAT ATT CAG GTA CGC AGA G Accession number AC003026 Exon 13
11927-11949 MRP1-E13f CAC TGC TCC TAG GAT GAT GAC TC A 312 bp
12238-12218 MRP1-E13r GAG TGT GAT CTA GAG GCT GCG Exon 14
15397-15419 MRP1-E14f GGG GAA ACC CTT GAA AGT TAA CC A 264 bp
15660-15638 MRP1-E14r CAG CCA AGG GAA AGA AAT GCA AG Exon 15
20044-20063 MRP1-E15f ATG CCT AGC GCC ATT CGT GC A 285 bp
20328-20309 MRP1-E15r GGG AGC ACG GTG GGA ATT CG Exon 16
23040-23063 MRP1-E16f GAA GGA ATG TTG AGG CCT TCA A 402 bp GTG
23441-23418 MRP1-E16r GAA AAG AGA CGT TGC TGC TTT CGC Exon 17
27108-27128 MRP1-E17f AAG TGA GGC CCT CCT AGC AGG C 372 bp
27479-27458 MRP1-E17r TGA TAG CAG CAG ACT CAC AGC C Exon 18
30588-30607 MRP1-E18f ACA CTC GGC CTG CTT CTA CG A 326 bp
30913-30892 MRP1-E18r AAG GAC TCC TAA AGG GGA CAC G Exon 19
34085-34105 MRP1-E19f GCT CCT GGA TGC TGT TAT CGC A 430 bp
34514-34495 MRP1-E19r2 TGG CTG GTG GCA ACC TCA AAG Accession number
AC003026 Exon 20 46405-46427 MR-E20f2 CCC TTG GTT TTA GCA TCT GCC
TC A 239 bp 46643-46621 MR-E20r GGG CTG AGG CCT TTT TTT GTT CC Exon
21 50449-50471 MRP1-E21f TGT GTG CAT GTG GAA ACA CTC CG A 368 bp
50816-50792 MRP1-E21r GAC AGG TGA GTT AAC ATA GAC AAG G Accession
number AC003026 Exon 22 55116-55134 MRP1-E22f TGC TGG TGA AGC CCC
CGA C A 402 bp 55517-55497 MRP1-E22r GTT TGG GGT CCC ACA AAA CGC
Exon 23 58530-58548 MRP1-E23f3 CTC CCT GCA GTG CCT GGT C A 474 bp
59003-58983 MRP1-E23r3 CCA CAC TGG GGA CAT GGT AAG Exon 24
65670-65688 MRP1-E24f1 AGG GCA GCC CGG CTC TAA C A 444 bp
66113-66093 MRP1-E24r GCC GGG GTT TGG CTT TAT ACC Accession number
U91318 Exon 25 4270-4292 MRP1-E25f CTC TCT CTG GAA TTA CTG CGG AG A
385 bp 4654-4634 MRP1-E25r CTG CTC CTC AAA CTC CGT ACC Exon 26
5371-5393 MRP1-E26f GAA AGT CAA GTA CGC CCG CTT AC A 242 bp
5612-5593 MRP1-E26r AGG TGC ACA GGA TAG GGT CC Exon 27 11200-11220
MRP1-E27f CTG AGA GGG TGC TCT GTA TCG A 545 bp 11744-11721
MRP1-E27r CAC TTC TGC AAG TTG TAT GCG CTC Exon 28 13844-13863
MR-E28f GAG AGG GCT GTC GAG TTG GG C 349 bp 14192-14170 MR-E28r TCA
GTG CAA TCA TAG GGC TTG CC Accession number U91318 Exon 29
16017-16036 MR-E29f CCA GAA GTC CTT AGG TCG CC A 317 bp 16333-16311
MR-E29r CTT CAA ACA CCC CTA CCG AGA TG Exon 30 17859-17880 MR-E30f
GGA CAT GCT TTC CTG GTC AAG C A 430 bp 18288-18268 MR-E30r GGG CTG
TCA CTA GGG ATA AGG Exon 31, (incl. 3'- 20650-20670 MR-E31f GCA ACC
AGC TGG AAG GTA CTG A 592 bp UTR) 21241-21219 MR-E31r CAG AAG TCT
GGC TGC CAA AAC TC Conditions for the different PCR fragments: PCRs
were carried out for all fragments in a reaction volume of 50
.mu.l. 40 ng DNA template was added to standard PCR buffer
containing 1.5 mM MgCl2 (Qiagen, Hilden), 200 .mu.M dNTP's (Roth,
Karlsruhe), 0.4 .mu.M (conditions A and C) or 1.6 .mu.M (condition
B) of each primer (Metabion, Munich), 10 .mu.l Q-Solution
(condition C; Qiagen, Hilden), 4 .mu.l DMSO (condition B) and 1 U
Taq polymerase (Qiagen, Hilden). All PCRs (conditions A and C) were
performed on a Perkin Elmer thermocycler (model 9700) with an
initial denaturation step of 2 min at 94.degree. C. and 34
amplification cycles of denaturation at 94.degree. C. for 45 sec,
primer annealing at 62.degree. C. for 45 sec, and 1 min for
72.degree. C. followed by a final extension of 72.degree. C. for 10
min. In the case of condition B the PCR reaction was performed with
an initial denaturation step of 3 min at 96.degree. C. and 35
amplification cycles of denaturation at 96.degree. C. for 45 sec,
primer annealing at 62.degree. C. for 30 sec, and 1 min for
72.degree. C. followed by a final extension of 72.degree. C. for 10
min.
[0177]
9TABLE 2 New SNP's in the gene for MRP1 Position of PCR fragment
the name variation wt-sequence wt/mut- and/or mut-sequence
Accession wt/mut: number AC026452 Exon 1/Prom 1 124667 f:
GCGTGCCCAGTCCTGGGGTTT f: GCGTGCCCAGT/CCCTGGGGTTT (intron 1) (SNP
34) (SEQ ID No: 071) (SEQ ID No: 073) r: AAACCCCAGGACTGGGCACGC r:
AAACCCCAGGA/GCTGGGCACGC (SEQ ID No: 072) (SEQ ID No: 074) mut/mut:
f: GCGTGCCCAGCCCTGGGGTTT (SEQ ID No: 075) r: AAACCCCAGGGCTGGGCACGC
(SEQ ID No: 076) Accession number U07050 wt/mut: Exon 1/Prom 1 1884
f: AGCCTTGGAGGATCTGGGGTG f: AGCCTTGGAGG/AATCTGGGGTG (SNP 33) (SEQ
ID No: 077) (SEQ ID No: 079) r: CACCCCAGATCCTCCAAGGCT r:
CACCCCAGATC/TCTCCAAGGCT (SEQ ID No: 078) (SEQ ID No: 080) mut/mut:
f: AGCCTTGGAGAATCTGGGGTG (SEQ ID No: 081) r: CACCCCAGATTCTCCAAGGCT
(SEQ ID No: 082) wt/mut: Promoter 1720-1723 f:
ACTCCAGGCAGGTAGGGGGCTCCG f: ACTCCAGGCAGGTA/delGGTAGGGGGCTCCG
fragment 2 del GGTA (SEQ ID No: 083) (SEQ ID No: 085) (SNP 25) r:
CGGAGCCCCCTACCTGCCTGGAGT r: CGGAGCCCCCTACC/delTACCTGCCTGGAGT (SEQ
ID No: 084) (SEQ ID No: 086) mut/mut: f:
ACTCCAGGCAdelGGTAGGGGGCTCCG (SEQ ID No: 087) r:
CGGAGCCCCCdelTACCTGCCTGGAGT (SEQ ID No: 088) Accession number
U07050 wt/mut: Promoter 1163 f: TGTGATCGGCCCGCCTCGGCT f:
TGTGATCGGCC/TCGCCTCGGCT fragment 3 (SNP 22) (SEQ ID No: 089) (SEQ
ID No: 091) r: AGCCGAGGCGGGCCGATCACA r: AGCCGAGGCGG/AGCCGATCACA
(SEQ ID No: 090) (SEQ ID No: 092) mut/mut: f: TGTGATCGGCTCGCCTCGGCT
(SEQ ID No: 093) r: AGCCGAGGCGAGCCGATCACA (SEQ ID No: 094) wt/mut:
Promoter 926 f: TTAATTTTTTTATTATTATTT f: TTAATTTTTTT/insTATTATTATTT
fragment 3 (SNP 21) (SEQ ID No: 095) (SEQ ID No: 097) r:
AAATAATAATAAAAAAATTAA r: AAATAATAATA/insAAAAAAATTAA (SEQ ID No:
096) (SEQ ID No: 098) mut/mut: f: TTAATTTTTTTinsTATTATTATTT (SEQ ID
No: 099) r: AAATAATAATinsAAAAAAAATTAA (SEQ ID No: 100) wt/mut:
Promoter 437 f: TTCCTCCTTCCCTCGCTAGGT f:
TTCCTCCTTCC/insTCCTTCCCTCGCTAGGT fragment 4 (SNP 31) (SEQ ID No:
101) (SEQ ID No: 103) r: ACCTAGCGAGGGAAGGAGGAA r:
ACCTAGCGAGG/insAGGAAGGGAAGGAGGAA (SEQ ID No: 102) (SEQ ID No: 104)
mut/mut: f: TTCCTCCTTCCTCCTTCCCTCGCTAGGT (SEQ ID No: 105) r:
ACCTAGCGAGGGAAGGAGGAAGGAGGAA (SEQ ID No: 106) Accession number
U07050 wt/mut: Promoter 381 f: TGGGGGACCCAGGCCAATAAA f:
TGGGGGACCCA/GGGCCAATAAA fragment 5 (SNP 20/30) (SEQ ID No: 107)
(SEQ ID No: 109) r: TTTATTGGCCTGGGTCCCCCA r:
TTTATTGGCCT/CGGGTCCCCCA (SEQ ID No: 108) (SEQ ID No: 110) mut/mut:
f: TGGGGGACCCGGGCCAATAAA (SEQ ID No: 111) r: TTTATTGGCCCGGGTCCCCCA
(SEQ ID No: 112) wt/mut: Promoter 233 f: AAGAGTAGCAGTTTTATCTTG f:
AAGAGTAGCAG/ATTTTATCTTG fragment 5 (SNP 19) (SEQ ID No: 113) (SEQ
ID No: 115) r: CAAGATAAAACTGCTACTCTT r: CAAGATAAAAC/TTGCTACTCTT
(SEQ ID No: 114) (SEQ ID No: 116) mut/mut: f: AAGAGTAGCAATTTTATCTTG
(SEQ ID No: 117) r: CAAGATAAAATTGCTACTCTT (SEQ ID No: 118) wt/mut:
Promoter 189 f: AAAAAAATCCCAATCCAAAAA f: AAAAAAATCCC/AAATCCAAAAA
fragment 5 (SNP 35) (SEQ ID No: 119) (SEQ ID No: 121) r:
TTTTTGGATTGGGATTTTTTT r: TTTTTGGATTG/TGGATTTTTTT (SEQ ID No: 120)
(SEQ ID No: 122) mut/mut: f: AAAAAAATCCAAATCCAAAAA (SEQ ID No: 123)
r: TTTTTGGATTTGGATTTTTTT (SEQ ID No: 124) GI number 7209451 wt/mut:
Exon 2 (intron 2) 39508 f: GTTTCGTTGTGGGGGGTGGGA f:
GTTTCGTTGTG/AGGGGGTGGGA (SNP 1) (SEQ ID No: 125) (SEQ ID No: 127)
r: TCCCACCCCCCACAACGAAAC r: TCCCACCCCCC/TACAACGAAAC (SEQ ID No:
126) (SEQ ID No: 128) mut/mut: f: GTTTCGTTGTAGGGGGTGGGA (SEQ ID No:
129) r: TCCCACCCCCTACAACGAAAC (SEQ ID No: 130) Accession number
AF022828 wt/mut: Exon 6 (intron 6) 174 f: CCAGGCCCCCCAGACCTCAGG f:
CCAGGCCCCCC/TAGACCTCAGG (SNP 10) (SEQ ID No: 131) (SEQ ID No: 133)
r: CCTGAGGTCTGGGGGGCCTGG r: CCTGAGGTCTG/AGGGGGCCTGG (SEQ ID No:
132) (SEQ ID No: 134) mut/mut: f: CCAGGCCCCCTAGACCTCAGG (SEQ ID No:
135) r: CCTGAGGTCTAGGGGGCCTGG (SEQ ID No: 136) Accession number
AF022829 wt/mut: Exon 7 (intron 7) 248 f: CCTTTCCACTCCTGTGGCCTC f:
CCTTTCCACTC/ACTGTGGCCTC (SNP 2) (SEQ ID No: 137) (SEQ ID No: 139)
r: GAGGCCACAGGAGTGGAAAGG r: GAGGCCACAGG/TAGTGGAAAGG (SEQ ID No:
138) (SEQ ID No: 140) mut/mut: f: CCTTTCCACTACTGTGGCCTC (SEQ ID No:
141) r: GAGGCCACAGTAGTGGAAAGG (SEQ ID No: 142) wt/mut: Exon 7
(intron 7) 258 f: CCTGTGGCCTCAATCCAGGAT f: CCTGTGGCCTC/GAATCCAGGAT
(SNP 3) (SEQ ID No: 143) (SEQ ID No: 145) r: ATCCTGGATTGAGGCCACAGG
r: ATCCTGGATTG/CAGGCCACAGG (SEQ ID No: 144) (SEQ ID No: 146)
mut/mut: f: CCTGTGGCCTGAATCCAGGAT (SEQ ID No: 147) r:
ATCCTGGATTCAGGCCACAGG (SEQ ID No: 148) Accession number AF022830
wt/mut: Exon 8 79 f: CCAGGCAGCCGGTGAAGGTTG f:
CCAGGCAGCCG/AGTGAAGGTTG (SNP 4) (SEQ ID No: 149) (SEQ ID No: 151)
r: CAACCTTCACCGGCTGCCTGG r: CAACCTTCACC/TGGCTGCCTGG (SEQ ID No:
150) (SEQ ID No: 152) mut/mut: f: CCAGGCAGCCAGTGAAGGTTG (SEQ ID No:
153) r: CAACCTTCACTGGCTGCCTGG (SEQ ID No: 154) Accession number
AF022830 wt/mut: Exon 8 88 f: CGGTGAAGGTTGTGTACTCCT f:
CGGTGAAGGTT/CGTGTACTCCT (SNP 5) (SEQ ID No: 155) (SEQ ID No: 157)
r: AGGAGTACACAACCTTCACCG r: AGGAGTACACA/GACCTTCACCG (SEQ ID No:
156) (SEQ ID No: 158) mut/mut: f: CGGTGAAGGTCGTGTACTCCT (SEQ ID No
159) r: AGGAGTACACGACCTTCACCG (SEQ ID No: 160) wt/mut: Exon 8 249
f: CTCATGAGCTTCTTCTTCAAG f: CTCATGAGCTT/GCTTCTTCAAG (SNP 37) (SEQ
ID No: 161) (SEQ ID No: 163) (only in r: CTTGAAGAAGAAGCTCATGAG r:
CTTGAAGAAGA/CAGCTCATGAG RCC (SEQ ID No: 162) (SEQ ID No: 164)
samples) mut/mut: f: CTCATGAGCTGCTTCTTCAAG (SEQ ID No: 165) r:
CTTGAAGAAGCAGCTCATGAG (SEQ ID No: 166) Accession number AF022831
wt/mut: Exon 9 95 f: AGTTCGTGAATGACACGAAGG f:
AGTTCGTGAAT/CGACACGAAGG (SNP 6) (SEQ ID No: 167) (SEQ ID No: 169)
r: CCTTCGTGTCATTCACGAACT r: CCTTCGTGTCA/GTTCACGAACT (SEQ ID No:
168) (SEQ ID No: 170) mut/mut: f: AGTTCGTGAACGACACGAAGG (SEQ ID No:
171) r: CCTTCGTGTCGTTCACGAACT (SEQ ID No: 172) wt/mut: Exon 9
(intron 9) 259 f: AAGGTAGGGGACGCTGTGCCA f: AAGGTAGGGGA/GCGCTGTGCCA
(SNP 7) (SEQ ID No: 173) (SEQ ID No: 175) r: TGGCACAGCGTCCCCTACCTT
r: TGGCACAGCGT/CCCCCTACCTT (SEQ ID No: 174) (SEQ ID No: 176)
mut/mut: f: AAGGTAGGGGGCGCTGTGCCA (SEQ ID No: 177) r:
TGGCACAGCGCCCCCTACCTT (SEQ ID No: 178) GI number 7209451 wt/mut:
Exon 10 57998 f: ACGCTCAGAGGTTCATGGACT f: ACGCTCAGAGG/TTTCATGGACT
(SNP 11) (SEQ ID No: 179) (SEQ ID No: 181) r: AGTCCATGAACCTCTGAGCGT
r: AGTCCATGAAC/ACTCTGAGCGT (SEQ ID No: 180) (SEQ ID No: 182)
mut/mut: f: ACGCTCAGAGTTTCATGGACT (SEQ ID No: 183) r:
AGTCCATGAAACTCTGAGCGT (SEQ ID No: 184) wt/mut: Exon 10 (intron
57853 f: GGCAGTGGGCCGAGGGAGTGG f: GGCAGTGGGCC/TGAGGGAGTGG 10) (SNP
8) (SEQ ID No: 185) (SEQ ID No: 187) r: CCACTCCCTCGGCCCACTGCC r:
CCACTCCCTCG/AGCCCACTGCC (SEQ ID No: 186) (SEQ ID No: 188) mut/mut:
f: GGCAGTGGGCTGAGGGAGTGG (SEQ ID No: 189) r: CCACTCCCTCAGCCCACTGCC
(SEQ ID No: 190) wt/mut: Exon 11 (intron 53282 f:
GCCAGTTGGACTCACTTGGGG f: GCCAGTTGGAC/GTCACTTGGGG 11) (SNP 12) (SEQ
ID No: 191) (SEQ ID No: 193) r: CCCCAAGTGAGTCCAACTGGC r:
CCCCAAGTGAG/CTCCAACTGGC (SEQ ID No: 192) (SEQ ID No: 194) mut/mut:
f: GCCAGTTGGAGTCACTTGGGG (SEQ ID No: 195) r: CCCCAAGTGACTCCAACTGGC
(SEQ ID No: 196) Accession number AC026452 wt/mut: Exon 13 (intron
137710 f: ACTCTCACTCAGGGCACAGCA f: ACTCTCACTCA/GGGGCACAGCA 12) (SNP
26) (SEQ ID No: 197) (SEQ ID No: 199) r: TGCTGTGCCCTGAGTGAGAGT r:
TGCTGTGCCCT/CGAGTGAGAGT (SEQ ID No: 198) (SEQ ID No: 200) mut/mut:
f: ACTCTCACTCGGGGCACAGCA (SEQ ID No: 201) r: TGCTGTGCCCCGAGTGAGAGT
(SEQ ID No: 202) Accession number AC026452 wt/mut: Exon 13 137667
f: GCAGGTGGCCCTGTGCACATT f: GCAGGTGGCCC/TTGTGCACATT (SNP 13) (SEQ
ID No: 203) (SEQ ID No: 205) r: AATGTGCACAGGGCCACCTGC r:
AATGTGCACAG/AGGCCACCTGC (SEQ ID No: 204) (SEQ ID No: 206) mut/mut:
f: GCAGGTGGCCTTGTGCACATT (SEQ ID No: 207) r: AATGTGCACAAGGCCACCTGC
(SEQ ID No: 208) wt/mut: Exon 13 137647 f: TTGCCGTCTACGTGACCATTG f:
TTGCCGTCTAC/TGTGACCATTG (SNP 14) (SEQ ID No: 209) (SEQ ID No: 211)
r: CAATGGTCACGTAGACGGCAA r: CAATGGTCACG/ATAGACGGCAA (SEQ ID No:
210) (SEQ ID No: 212) mut/mut: f: TTGCCGTCTATGTGACCATTG (SEQ ID No:
213) r: CAATGGTCACATAGACGGCAA (SEQ ID No: 214) Accession wt/mut:
number AC003026 Exon 17 (intron 27159 f: TCGTTGATCAGATCTGTCTGT f:
TCGTTGATCAG/CATCTGTCTGT 16) (SNP: mr-v-024) (SEQ ID No: 215) (SEQ
ID No: 217) r: ACAGACAGATCTGATCAACGA r: ACAGACAGATC/GTGATCAACGA
(SEQ ID No: 216) (SEQ ID No: 218) mut/mut: f: TCGTTGATCACATCTGTCTGT
(SEQ ID No: 219) r: ACAGACAGATGTGATCAACGA (SEQ ID No: 220) wt/mut:
Exon 17 27258 f: GATTCTCTCCGAGAAAACATC f: GATTCTCTCCG/AAGAAAACATC
(SNP 9) (SEQ ID No: 221) (SEQ ID No: 223) r: GATGTTTTCTCGGAGAGAATC
r: GATGTTTTCTC/TGGAGAGAATC (SEQ ID No: 222) (SEQ ID No: 224)
mut/mut: f: GATTCTCTCCAAGAAAACATC (SEQ ID No: 225) r:
GATGTTTTCTTGGAGAGAATC (SEQ ID No: 226) Accession number AC003026
wt/mut: Exon 19 (intron 34206/34207 f: AGTCTCACACATGTGCACTCAC f:
AGTCTCACACAT/delATGTGCACTCAC 18) (SNP 18) (SEQ ID No: 227) (SEQ ID
No: 229) r: GTGAGTGCACATGTGTGAGACT r: GTGAGTGCACAT/delATGTGTGAGACT
(SEQ ID No: 228) (SEQ ID No: 230) mut/mut: f:
AGTCTCACACdelATGTGCACTCAC (SEQ ID No: 231) r:
GTGAGTGCACdelATGTGTGAGACT (SEQ ID No: 232) wt/mut: Exon 19 (intron
34215 f: CATGTGCACTGACGTGGCCGG f: CATGTGCACTG/CACGTGGCCGG 18) (SNP
17) (SEQ ID No: 233) (SEQ ID No: 235) r: CCGGCCACGTCAGTGCACATG r:
CCGGCCACGTC/GAGTGCACATG (SEQ ID No: 234) (SEQ ID No: 236) mut/mut:
f: CATGTGCACTCACGTGGCCGG (SEQ ID No: 237) r: CCGGCCACGTGAGTGCACATG
(SEQ ID No: 238) wt/mut: Exon 22 (intron 55156 f:
GGGGCTGGGGCTGGGTGCGTG f: GGGGCTGGGGC/insTGGGGCTGGGTGCGTG 21) (SNP
28) (SEQ ID No: 239) (SEQ ID No: 241) r: CACGCACCCAGCCCCAGCCCC r:
CACGCACCCAG/insGCCCCACCCCAGCCCC (SEQ ID No: 240) (SEQ ID No: 242)
mut/mut: f: GGGGCTGGGGCinsTGGGGCTGGGTGCGTG (SEQ ID No: 243) r:
CACGCACCCAinsGCCCCAGCCCCAGCCCC (SEQ ID No: 244) Accession wt/mut:
number AC003026 Exon 22 (intron 55472 f: TGTCTAATTATAGAAATGGAT f:
TGTCTAATTAT/CAGAAATGGAT 22) (SNP 27) (SEQ ID No: 245) (SEQ ID No:
247) r: ATCCATTTCTATAATTAGACA r: ATCCATTTCTA/GTAATTAGACA (SEQ ID
No: 246) (SEQ ID No: 248) mut/mut: f: TGTCTAATTACAGAAATGGAT (SEQ ID
No: 249) r: ATCCATTTCTGTAATTAGACA (SEQ ID No: 250) Accession number
U91318 wt/mut: Exon 28 14008 f: CTGGGAAGTCGTCCCTGACCC f:
CTGGGAAGTCG/ATCCCTGACCC (SNP 23) (SEQ ID No: 251) (SEQ ID No: 253)
r: GGGTCAGGGACGACTTCCCAG r: GGGTCAGGGAC/TGACTTCCCAG (SEQ ID No:
252) (SEQ ID No: 254) mut/mut: f: CTGGGAAGTCATCCCTGACCC (SEQ ID No:
255) r: GGGTCAGGGATGACTTCCCAG (SEQ ID No: 256) Accession number
AC025277 wt/mut: Exon 29 (intron 150727 f: CCATGTCAGCGTGACACAGGT f:
CCATGTCAGCG/ATGACACAGGT 28) (SNP 24) (SEQ ID No: 257) (SEQ ID No:
259) r: ACCTGTGTCACGCTGACATGG r: ACCTGTGTCAC/TGCTGACATGG (SEQ ID
No: 258) (SEQ ID No: 260) mut/mut: f: CCATGTCAGCATGACACAGGT (SEQ ID
No: 261) r: ACCTGTGTCATGCTGACATGG (SEQ ID No: 262) Accession number
U91318 wt/mut: Exon 30 (intron 17970 f: CTGGTTTTTTTCTTCCGGTCA f:
CTGGTTTTTTT/delTCTTCCGGTCA 29) (SNP 15) (SEQ ID No: 263) (SEQ ID
No: 265) r: TGACCGGAAGAAAAAAACCAG r: TGACCGGAAGA/delAAAAAAACCAG
(SEQ ID No: 264) (SEQ ID No: 266) mut/mut: f:
CTGGTTTTTTdelTCTTCCGGTCA (SEQ ID No: 267) r:
TGACCGGAAGdelAAAAAAACCAG (SEQ ID No: 268) Accession number U91318
wt/mut: Exon 30 (intron 18195 f: CACTGGCACAGTGGCCTCTAG f:
CACTGGCACAG/ATGGCCTCTAG 30) (SNP 16) (SEQ ID No: 269) (SEQ ID No:
271) r: CTAGAGGCCACTGTGCCAGTG r: CTAGAGGCCAC/TTGTGCCAGTG (SEQ ID
No: 270) (SEQ ID No: 272) mut/mut: f: CACTGGCACAATGGCCTCTAG (SEQ ID
No: 273) r: CTAGAGGCCATTGTGCCAGTG (SEQ ID No: 274) wt/mut: Exon 31
(3' 21133 f: CCCAAAACACGCACACCCTGC f: CCCAAAACACG/ACACACCCTGC
flanking region) (SNP 29) (SEQ ID No: 275) (SEQ ID No: 277) r:
GCAGGGTGTGCGTGTTTTGGG r: GCAGGGTGTGC/TGTGTTTTGGG (SEQ ID No: 276)
(SEQ ID No: 278) mut/mut: f: CCCAAAACACACACACCCTGC (SEQ ID No: 279)
r: GCAGGGTGTGTGTGTTTTGGG (SEQ ID No: 280) Accession number AC003026
wt/mut: Exon 19 (intron 34218 f: GTGCACTCACGTGGCCGGGTG f:
GTGCACTCACG/ATGGCCGGGTG 18) (SNP 38)
(SEQ ID No: 281) (SEQ ID No: 283) (only in r: CACCCGGCCACGTGAGTGCAC
r: CACCCGGCCAC/TGTGAGTGCAC RCC (SEQ ID No: 282) (SEQ ID No: 284)
samples) mut/mut: f: GTGCACTCACATGGCCGGGTG (SEQ ID No: 285) r:
CACCCGGCCATGTGAGTGCAC (SEQ ID No: 286) Accession number U91318
wt/mut: Exon 30 18067 f: CCACGGCAGCCGTGGACCTGG f:
CCACGGCAGCC/TGTGGACCTGG (SNP 39) (SEQ ID No: 287) (SEQ ID No: 289)
(only in r: CCAGGTCCACGGCTGCCGTGG r: CCAGGTCCACG/AGCTGCCGTGG RCC
(SEQ ID No: 288) (SEQ ID No: 290) samples) mut/mut: f:
CCACGGCAGCTGTGGACCTGG (SEQ ID No: 291) r: CCAGGTCCACAGCTGCCGTGG
(SEQ ID No: 292) Accession number U07050 wt/mut: Promoter 440 f:
CTCCTTCCCTCGCTAGGTCCT f: CTCCTTCCCTC/TGCTAGGTCCT fragment 5 (SNP
40) (SEQ ID No: 293) (SEQ ID No: 295) (only in r:
AGGACCTAGCGAGGGAAGGAG r: AGGACCTAGCG/AAGGGAAGGAG RCC (SEQ ID No:
294) (SEQ ID No: 296) samples) mut/mut: f: CTCCTTCCCTTGCTAGGTCCT
(SEQ ID No: 297) r: AGGACCTAGCAAGGGAAGGAG (SEQ ID No: 298) wt/mut:
Promoter 1625 f: GGGAATCACTCAACCTCTCTG f: GGGAATCACTC/AAACCTCTCTG
fragment 2 (SNP 41) (SEQ ID No: 299) (SEQ ID No: 301) (only in r:
CAGAGAGGTTGAGTGATTCCC r: CAGAGAGGTTG/TAGTGATTCCC RCC (SEQ ID No:
300) (SEQ ID No: 302) samples) mut/mut: f: GGGAATCACTAAACCTCTCTG
(SEQ ID No: 303) r: CAGAGAGGTTTAGTGATTCCC (SEQ ID No: 304)
Accession number U91318 wt/mut: Exon 30 (intron 17900 f:
TGTCTCCTTTCGCTTCTCCCA f: TGTCTCCTTTC/TGCTTCTCCCA 29) (SNP 42) (SEQ
ID No: 305) (SEQ ID No: 307) (only in r: TGGGAGAAGCGAAAGGAGACA r:
TGGGAGAAGCG/AAAAGGAGACA RCC (SEQ ID No: 306) (SEQ ID No: 308)
samples) mut/mut: f: TGTCTCCTTTTGCTTCTCCCA (SEQ ID No: 309) r:
TGGGAGAAGCAAAAGGAGACA (SEQ ID No: 310) Accession number AC026452
wt/mut: Promoter 38646 f: CCTTAAACAGGATTTGAAAAG f:
CCTTAAACAGG/CATTTGAAAAG fragment 1 (SNP 32) (SEQ ID No: 311) (SEQ
ID No: 313) r: CTTTTCAAATCCTGTTTAAGG r: CTTTTCAAATC/GCTGTTTAAGG
(SEQ ID No: 312) (SEQ ID No: 314) mut/mut: f: CCTTAAACAGCATTTGAAAAG
(SEQ ID No: 315) r: CTTTTCAAATGCTGTTTAAGG (SEQ ID No. 316)
Accession number AC025277 wt/mut: Exon 5 (intron 5) 33551 f:
TGTGACCACAGATGAGTGTGT f: TGTGACCACAG/AATGAGTGTGT (SNP 36) (SEQ ID
No: 317) (SEQ ID No: 319) r: ACACACTCATCTGTGGTCACA r:
ACACACTCATC/TTGTGGTCACA (SEQ ID No: 318) (SEQ ID No: 320) mut/mut:
f: TGTGACCACAAATGAGTGTGT (SEQ ID No: 321) r: ACACACTCATTTGTGGTCACA
(SEQ ID No: 322)
[0178]
10TABLE 3 New SNP's in the gene for MRP1 GI no Seq Seq Seq Seq Site
SNP Var. Pos. Acc no ID Forward.sup.1 ID Reverse.sup.1 ID
IUB_Forward ID IUB_Reverse P3 mrys546 a > g 51798 3582311 329
TAACCAGGTTgT 330 GAGGATCAAcA 331 TAACCAGGTTrT 332 GAGGATCAAyA
TGATCCTC ACCTGGTTA TGATCCTC ACCTGGTTA P1 mryp282 g > a 37971
7363401 333 TGGGGTGGGGa 334 CCCCGCGCCAt 335 TGGGGTGGGGr 336
CCCCGCGCCAy TGGCGCGGGG CCCCACCCCA TGGCGCGGGG CCCCACCCCA P1 mryp877
g > a 50892 3582311 337 TGGGCACGCGa 338 TGCGTGGGGG 339
TGGGCACGCGr 340 TGCGTGGGGG CCCCCCACGCA GtCGCGTGCCCA CCCCCCACGCA
GyCGCGTGCCCA E22 mryo336 g > a 55296 2815549 341 CCATGTGTCCa 342
GAAGCCAGCGt 343 CCATGTGTCCrC 344 GAAGCCAGCGy CGCTGGCTTC GGACACATGG
GCTGGCTTC GGACACATGG I21 mryo172 g > a 55132 2815549 345
TGAAGCCCCCa 346 CCCACAAGGTtG 347 TGAAGCCCCCrA 348 CCCACAAGGTy
ACCTTGTGGG GGGGCTTCA CCTTGTGGG GGGGGCTTCA I21 mryo154 a > g
55114 2815549 349 TGGGTGGCACg 350 TCACCAGCACc 351 TGGGTGGCACr 352
TCACCAGCACy GTGCTGGTGA GTGCCACCCA GTGCTGGTGA GTGCCACCCA I21 mryo152
a > g 55112 2815549 353 GCTGGGTGGCg 354 ACCAAGCACTG 355
GCTGGGTGGCr 356 ACCAAGCACTG CAGTGCTGGT cGCCACCCAGC CAGTGCTGGT
yGCCACCCAGC P1 mryp522 delCCCG 109 4826837 357 GGCCCGATCAC 358
CGGCGGCGGG 359 GGCCCGATCAn 360 CGGCGGCGGG CCGCCC to CCGCCGCCG
TGATCGGGCC CCCGCCGCCG nTGATCGGGCC GGTG 122 P1 mryp491 delGCC 76
4826837 361 TCCCTGC[GCC] 362 CGCTAGCGCT 363 TCCCTGC[GCC] 364
CGCTAGCGCTn to .sub.13AGCGCTAGCG [GGC].sub.13GCAGGG
.sub.13nAGCGCTAGCG [GGC].sub.13GCAGGG 78 A A P1 mryp489
del[GCC].sub.2 73 4826837 365 TCCCTGC[GCC] 366 CGCTAGCGCT 367
TCCCTGC[GCC] 368 CGCTAGCGCTn to .sub.12AGCGCTAGCG
[GGC].sub.12GCAGGG .sub.12nAGCGCTAGCG [GGC].sub.12GCAGGG 78 A A P1
mryp486 del[GCC].sub.3 70 4826837 369 TCCCTGC[GCC] 370 CGCTAGCGCT
371 TCCCTGC[GCC] 372 CGCTAGCGCTn to .sub.11 AGCGCTAGCG
[GGC].sub.11GCAGGG .sub.11nAGCGCTAGCG [GGC].sub.11GCAGGG 78 A A P1
mryp483 del[GCC].sub.4 67 4826837 373 TCCCTGC[GCC] 374 CGCTAGCGCT
375 TCCCTGC[GCC] 376 CGCTAGCGCTn to .sub.10AGCGCTAGCG
[GGC].sub.10GCAGGG .sub.10nAGCGCTAGCG [GGC].sub.10GCAGGG 78 A A P1
mryp474 del[GCC].sub.7 58 4826837 377 TCCCTGC[GCC] 378 CGCTAGCGCT
379 TCCCTGC[GCC] 380 CGCTAGCGCTn to .sub.7AGCGCTAGCG
[GGC].sub.7GCAGGGA .sub.7nAGCGCTAGCG [GGC].sub.7GCAGGGA 78 I14
mrzl154 delAA 20097 2815549 381 TCAAGCAGAGA 382 AACACTCTCTCT 383
TCAAGCAGAGn 384 AACACTCTCTnC to GAGAGTGTT CTGCTTGA AGAGAGTGTT
TCTGCTTGA 20098 E9 mrzr176 c > t 60357 7209451 385 CTGGGGCCTTt
386 TGAATGACACaA 387 CTGGGGCCTTy 388 GAATGACACrA GTGTCATTCA
AGGCCCCAG GTGTCATTCA AGGCCCCAG I7 mrzs129 g > a 61786 7209451
389 ACACAAGGAGa 390 AACGGCTTCAtC 391 ACACAAGGAGrT 392 AACGGCTTCAy
TGAAGCCGTT TCCTTGTGT GAAGCCGTT CTCCTTGTGT I6 mrzu272 insC 76437/
7209451 393 CAGGCCCCCCc 394 CCTGAGGTCTg 395 CAGGCCCCCCn 396
CCTGAGGTCTn 76438 AGACCTCAGG GGGGGGCCTG AGACCTCAGG GGGGGGCCTG E2
mrzy349 g > a 39541 7209451 397 TACAGTTTTGaT 398 CTCAACAAAAtC
399 TACAGTTTTGrT 400 CTCAACAAAAyC TTTGTTGAG AAAACTGTA TTTGTTGAG
AAAACTGTA .sup.1Brackets depict repeats. Numbers indicate how often
the sequence in brackets is repeated.
[0179]
11TABLE 4 AAexchange ProtAccNo SeqID Protein mut SeqID Protein T731
GI:2828206 401 TPLNKiKTALG 402 TPLNKxKTALG A989T GI:2828206 403
CNHVStLASNY 404 CNHVSxLASNY
[0180]
Sequence CWU 1
1
406 1 19 DNA Artificial Sequence Description of Artificial Sequence
Primer 1 gtagggggct ccgttcacg 19 2 25 DNA Artificial Sequence
Description of Artificial Sequence Primer 2 cctggaaggt tgtttttaca
gacgg 25 3 19 DNA Artificial Sequence Description of Artificial
Sequence Primer 3 tggagactgg cgccgtctg 19 4 22 DNA Artificial
Sequence Description of Artificial Sequence Primer 4 aaggacagta
tccgtcacca gg 22 5 22 DNA Artificial Sequence Description of
Artificial Sequence Primer 5 catggggttg tgaggattgc ac 22 6 23 DNA
Artificial Sequence Description of Artificial Sequence Primer 6
tgagattcaa acccgtgagc agc 23 7 24 DNA Artificial Sequence
Description of Artificial Sequence Primer 7 cttagaaact cattcaccct
tggg 24 8 22 DNA Artificial Sequence Description of Artificial
Sequence Primer 8 gtgacaaggc ttcctaaggc tg 22 9 27 DNA Artificial
Sequence Description of Artificial Sequence Primer 9 gattaacatc
tgccatctta ccataag 27 10 21 DNA Artificial Sequence Description of
Artificial Sequence Primer 10 cctcccccca atcaaaggac c 21 11 21 DNA
Artificial Sequence Description of Artificial Sequence Primer 11
agctggtttc atgctccagg c 21 12 25 DNA Artificial Sequence
Description of Artificial Sequence Primer 12 ctagaagaag gaacttaggg
tcaac 25 13 21 DNA Artificial Sequence Description of Artificial
Sequence Primer 13 ttccagggcg gtctgttgta g 21 14 23 DNA Artificial
Sequence Description of Artificial Sequence Primer 14 attacttttg
gtctccactg agc 23 15 23 DNA Artificial Sequence Description of
Artificial Sequence Primer 15 aaaacccaac aactcctgtc ttg 23 16 20
DNA Artificial Sequence Description of Artificial Sequence Primer
16 gcatctttcc ctccgggtcc 20 17 21 DNA Artificial Sequence
Description of Artificial Sequence Primer 17 acccagcccc agaatgtgat
c 21 18 22 DNA Artificial Sequence Description of Artificial
Sequence Primer 18 gcacacacac tcatttgtgg tc 22 19 23 DNA Artificial
Sequence Description of Artificial Sequence Primer 19 gagcagctga
ctacttgcta agc 23 20 23 DNA Artificial Sequence Description of
Artificial Sequence Primer 20 cattcattca ttcactcccc acc 23 21 25
DNA Artificial Sequence Description of Artificial Sequence Primer
21 ctgtcattga ctctcattgc ctaac 25 22 21 DNA Artificial Sequence
Description of Artificial Sequence Primer 22 agtaacaggc agcactgcca
g 21 23 21 DNA Artificial Sequence Description of Artificial
Sequence Primer 23 atctctggca gaccccacaa c 21 24 24 DNA Artificial
Sequence Description of Artificial Sequence Primer 24 aactgaaaga
tcaaagccaa ggag 24 25 22 DNA Artificial Sequence Description of
Artificial Sequence Primer 25 ccccacgtgt cacaagtcat tc 22 26 20 DNA
Artificial Sequence Description of Artificial Sequence Primer 26
tgggctggaa atccccacgc 20 27 20 DNA Artificial Sequence Description
of Artificial Sequence Primer 27 gggaggagga gagatctgcg 20 28 20 DNA
Artificial Sequence Description of Artificial Sequence Primer 28
tgaaccacag ccggaactgc 20 29 20 DNA Artificial Sequence Description
of Artificial Sequence Primer 29 ggatggatca accggggaag 20 30 23 DNA
Artificial Sequence Description of Artificial Sequence Primer 30
tcagaatccc agatatgcag ccg 23 31 22 DNA Artificial Sequence
Description of Artificial Sequence Primer 31 tgttgagtga tgggctgatc
cc 22 32 28 DNA Artificial Sequence Description of Artificial
Sequence Primer 32 ccttttaaaa atattcaggt acgcagag 28 33 23 DNA
Artificial Sequence Description of Artificial Sequence Primer 33
cactgctcct aggatgatga ctc 23 34 21 DNA Artificial Sequence
Description of Artificial Sequence Primer 34 gagtgtgatc tagaggctgc
g 21 35 23 DNA Artificial Sequence Description of Artificial
Sequence Primer 35 ggggaaaccc ttgaaagtta acc 23 36 23 DNA
Artificial Sequence Description of Artificial Sequence Primer 36
cagccaaggg aaagaaatgc aag 23 37 20 DNA Artificial Sequence
Description of Artificial Sequence Primer 37 atgcctagcg ccattcgtgc
20 38 20 DNA Artificial Sequence Description of Artificial Sequence
Primer 38 gggagcacgg tgggaattcg 20 39 24 DNA Artificial Sequence
Description of Artificial Sequence Primer 39 gaaggaatgt tgaggccttc
agtg 24 40 24 DNA Artificial Sequence Description of Artificial
Sequence Primer 40 gaaaagagac gttgctgctt tcgc 24 41 21 DNA
Artificial Sequence Description of Artificial Sequence Primer 41
aagtgaggcc ctcctagcag g 21 42 22 DNA Artificial Sequence
Description of Artificial Sequence Primer 42 tgatagcagc agactcacag
cc 22 43 20 DNA Artificial Sequence Description of Artificial
Sequence Primer 43 acactcggcc tgcttctacg 20 44 22 DNA Artificial
Sequence Description of Artificial Sequence Primer 44 aaggactcct
aaaggggaca cg 22 45 21 DNA Artificial Sequence Description of
Artificial Sequence Primer 45 gctcctggat gctgttatcg c 21 46 21 DNA
Artificial Sequence Description of Artificial Sequence Primer 46
tggctggtgg caacctcaaa g 21 47 23 DNA Artificial Sequence
Description of Artificial Sequence Primer 47 cccttggttt tagcatctgc
ctc 23 48 23 DNA Artificial Sequence Description of Artificial
Sequence Primer 48 gggctgaggc ctttttttgt tcc 23 49 23 DNA
Artificial Sequence Description of Artificial Sequence Primer 49
tgtgtgcatg tggaaacact ccg 23 50 25 DNA Artificial Sequence
Description of Artificial Sequence Primer 50 gacaggtgag ttaacataga
caagg 25 51 19 DNA Artificial Sequence Description of Artificial
Sequence Primer 51 tgctggtgaa gcccccgac 19 52 21 DNA Artificial
Sequence Description of Artificial Sequence Primer 52 gtttggggtc
ccacaaaacg c 21 53 19 DNA Artificial Sequence Description of
Artificial Sequence Primer 53 ctccctgcag tgcctggtc 19 54 21 DNA
Artificial Sequence Description of Artificial Sequence Primer 54
ccacactggg gacatggtaa g 21 55 19 DNA Artificial Sequence
Description of Artificial Sequence Primer 55 agggcagccc ggctctaac
19 56 21 DNA Artificial Sequence Description of Artificial Sequence
Primer 56 gccggggttt ggctttatac c 21 57 23 DNA Artificial Sequence
Description of Artificial Sequence Primer 57 ctctctctgg aattactgcg
gag 23 58 21 DNA Artificial Sequence Description of Artificial
Sequence Primer 58 ctgctcctca aactccgtac c 21 59 23 DNA Artificial
Sequence Description of Artificial Sequence Primer 59 gaaagtcaag
tacgcccgct tac 23 60 20 DNA Artificial Sequence Description of
Artificial Sequence Primer 60 aggtgcacag gatagggtcc 20 61 21 DNA
Artificial Sequence Description of Artificial Sequence Primer 61
ctgagagggt gctctgtatc g 21 62 24 DNA Artificial Sequence
Description of Artificial Sequence Primer 62 cacttctgca agttgtatgc
gctc 24 63 20 DNA Artificial Sequence Description of Artificial
Sequence Primer 63 gagagggctg tcgagttggg 20 64 23 DNA Artificial
Sequence Description of Artificial Sequence Primer 64 tcagtgcaat
catagggctt gcc 23 65 20 DNA Artificial Sequence Description of
Artificial Sequence Primer 65 ccagaagtcc ttaggtcgcc 20 66 23 DNA
Artificial Sequence Description of Artificial Sequence Primer 66
cttcaaacac ccctaccgag atg 23 67 22 DNA Artificial Sequence
Description of Artificial Sequence Primer 67 ggacatgctt tcctggtcaa
gc 22 68 21 DNA Artificial Sequence Description of Artificial
Sequence Primer 68 gggctgtcac tagggataag g 21 69 21 DNA Artificial
Sequence Description of Artificial Sequence Primer 69 gcaaccagct
ggaaggtact g 21 70 23 DNA Artificial Sequence Description of
Artificial Sequence Primer 70 cagaagtctg gctgccaaaa ctc 23 71 21
DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide 71 gcgtgcccag tcctggggtt t 21 72 21 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide 72 aaaccccagg actgggcacg c 21 73 21 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 73 gcgtgcccag ycctggggtt t 21 74 21 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 74 aaaccccagg rctgggcacg c 21 75 21 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 75 gcgtgcccag ccctggggtt t 21 76 21 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 76 aaaccccagg gctgggcacg c 21 77 21 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 77 agccttggag gatctggggt g 21 78 21 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 78 caccccagat cctccaaggc t 21 79 21 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 79 agccttggag ratctggggt g 21 80 21 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 80 caccccagat yctccaaggc t 21 81 21 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 81 agccttggag aatctggggt g 21 82 21 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 82 caccccagat tctccaaggc t 21 83 24 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 83 actccaggca ggtagggggc tccg 24 84 24 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide 84 cggagccccc tacctgcctg gagt 24 85 24 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide 85 actccaggca ggtagggggc tccg 24 86 24 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide 86 cggagccccc tacctgcctg gagt 24 87 20 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide 87 actccaggca gggggctccg 20 88 20 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 88 cggagccccc tgcctggagt 20 89 21 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 89 tgtgatcggc ccgcctcggc t 21 90 21 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 90 agccgaggcg ggccgatcac a 21 91 21 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 91 tgtgatcggc ycgcctcggc t 21 92 21 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 92 agccgaggcg rgccgatcac a 21 93 21 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 93 tgtgatcggc tcgcctcggc t 21 94 21 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 94 agccgaggcg agccgatcac a 21 95 21 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 95 ttaatttttt tattattatt t 21 96 21 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 96 aaataataat aaaaaaatta a 21 97 22 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 97 ttaatttttt ttattattat tt 22 98 22 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 98 aaataataat aaaaaaaatt aa 22 99 22 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 99 ttaatttttt ttattattat tt 22 100 22 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide 100 aaataataat aaaaaaaatt aa 22 101 21 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide 101 ttcctccttc cctcgctagg t 21 102 21 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide 102 acctagcgag ggaaggagga a 21 103 28 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide 103 ttcctccttc ctccttccct cgctaggt 28 104 28 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide 104 acctagcgag gaggaaggga aggaggaa 28 105 28 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide 105 ttcctccttc ctccttccct cgctaggt 28 106 28 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide 106 acctagcgag ggaaggagga aggaggaa 28 107 21 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide 107 tgggggaccc aggccaataa a 21 108 21 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide 108 tttattggcc tgggtccccc a 21 109 21 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide 109 tgggggaccc rggccaataa a 21 110 21 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide 110 tttattggcc ygggtccccc a 21 111 21 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide 111 tgggggaccc gggccaataa a 21 112 21 DNA
Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 112 tttattggcc
cgggtccccc a 21 113 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 113 aagagtagca
gttttatctt g 21 114 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 114 caagataaaa
ctgctactct t 21 115 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 115 aagagtagca
rttttatctt g 21 116 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 116 caagataaaa
ytgctactct t 21 117 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 117 aagagtagca
attttatctt g 21 118 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 118 caagataaaa
ttgctactct t 21 119 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 119 aaaaaaatcc
caatccaaaa a 21 120 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 120 tttttggatt
gggatttttt t 21 121 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 121 aaaaaaatcc
maatccaaaa a 21 122 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 122 tttttggatt
kggatttttt t 21 123 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 123 aaaaaaatcc
aaatccaaaa a 21 124 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 124 tttttggatt
tggatttttt t 21 125 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 125 gtttcgttgt
ggggggtggg a 21 126 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 126 tcccaccccc
cacaacgaaa c 21 127 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 127 gtttcgttgt
rgggggtggg a 21 128 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 128 tcccaccccc
yacaacgaaa c 21 129 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 129 gtttcgttgt
agggggtggg a 21 130 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 130 tcccaccccc
tacaacgaaa c 21 131 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 131 ccaggccccc
cagacctcag g 21 132 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 132 cctgaggtct
ggggggcctg g 21 133 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 133 ccaggccccc
yagacctcag g 21 134 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 134 cctgaggtct
rgggggcctg g 21 135 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 135 ccaggccccc
tagacctcag g 21 136 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 136 cctgaggtct
agggggcctg g 21 137 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 137 cctttccact
cctgtggcct c 21 138 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 138 gaggccacag
gagtggaaag g 21 139 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 139 cctttccact
mctgtggcct c 21 140 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 140 gaggccacag
kagtggaaag g 21 141 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 141 cctttccact
actgtggcct c 21 142 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 142 gaggccacag
tagtggaaag g 21 143 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 143 cctgtggcct
caatccagga t 21 144 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 144 atcctggatt
gaggccacag g 21 145 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 145 cctgtggcct
saatccagga t 21 146 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 146 atcctggatt
saggccacag g 21 147 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 147 cctgtggcct
gaatccagga t 21 148 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 148 atcctggatt
caggccacag g 21 149 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 149 ccaggcagcc
ggtgaaggtt g 21 150 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 150 caaccttcac
cggctgcctg g 21 151 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 151 ccaggcagcc
rgtgaaggtt g 21 152 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 152 caaccttcac
yggctgcctg g 21 153 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 153 ccaggcagcc
agtgaaggtt g 21 154 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 154 caaccttcac
tggctgcctg g 21 155 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 155 cggtgaaggt
tgtgtactcc t 21 156 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 156 aggagtacac
aaccttcacc g 21 157 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 157 cggtgaaggt
ygtgtactcc t 21 158 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 158 aggagtacac
raccttcacc g 21 159 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 159 cggtgaaggt
cgtgtactcc t 21 160 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 160 aggagtacac
gaccttcacc g 21 161 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 161 ctcatgagct
tcttcttcaa g 21 162 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 162 cttgaagaag
aagctcatga g 21 163 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 163 ctcatgagct
kcttcttcaa g 21 164 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 164 cttgaagaag
magctcatga g 21 165 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 165 ctcatgagct
gcttcttcaa g 21 166 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 166 cttgaagaag
cagctcatga g 21 167 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 167 agttcgtgaa
tgacacgaag g 21 168 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 168 ccttcgtgtc
attcacgaac t 21 169 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 169 agttcgtgaa
ygacacgaag g 21 170 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 170 ccttcgtgtc
rttcacgaac t 21 171 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 171 agttcgtgaa
cgacacgaag g 21 172 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 172 ccttcgtgtc
gttcacgaac t 21 173 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 173 aaggtagggg
acgctgtgcc a 21 174 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 174 tggcacagcg
tcccctacct t 21 175 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 175 aaggtagggg
rcgctgtgcc a 21 176 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 176 tggcacagcg
ycccctacct t 21 177 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 177 aaggtagggg
gcgctgtgcc a 21 178 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 178 tggcacagcg
ccccctacct t 21 179 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 179 acgctcagag
gttcatggac t 21 180 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 180 agtccatgaa
cctctgagcg t 21 181 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 181 acgctcagag
kttcatggac t 21 182 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 182 agtccatgaa
mctctgagcg t 21 183 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 183 acgctcagag
tttcatggac t 21 184 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 184 agtccatgaa
actctgagcg t 21 185 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 185 ggcagtgggc
cgagggagtg g 21 186 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 186 ccactccctc
ggcccactgc c 21 187 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 187 ggcagtgggc
ygagggagtg g 21 188 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 188 ccactccctc
rgcccactgc c 21 189 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 189 ggcagtgggc
tgagggagtg g 21 190 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 190 ccactccctc
agcccactgc c 21 191 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 191 gccagttgga
ctcacttggg g 21 192 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 192 ccccaagtga
gtccaactgg c 21 193 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 193 gccagttgga
stcacttggg g 21 194 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 194 ccccaagtga
stccaactgg c 21 195 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 195 gccagttgga
gtcacttggg g 21 196 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 196 ccccaagtga
ctccaactgg c 21 197 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 197 actctcactc
agggcacagc a 21 198 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 198 tgctgtgccc
tgagtgagag t 21 199 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 199 actctcactc
rgggcacagc a 21 200 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 200 tgctgtgccc
ygagtgagag t 21 201 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 201 actctcactc
ggggcacagc a 21 202 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 202 tgctgtgccc
cgagtgagag t 21 203 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 203 gcaggtggcc
ctgtgcacat t 21 204 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 204 aatgtgcaca
gggccacctg c 21 205 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 205 gcaggtggcc
ytgtgcacat t 21 206 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 206 aatgtgcaca
rggccacctg c 21 207 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 207 gcaggtggcc
ttgtgcacat t 21 208 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 208 aatgtgcaca
aggccacctg c 21 209 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 209 ttgccgtcta
cgtgaccatt g 21 210 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 210 caatggtcac
gtagacggca a 21 211 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 211 ttgccgtcta
ygtgaccatt g 21 212 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 212 caatggtcac
rtagacggca a 21 213 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 213 ttgccgtcta
tgtgaccatt g 21 214 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 214 caatggtcac
atagacggca a 21 215 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 215 tcgttgatca
gatctgtctg t 21 216 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 216 acagacagat
ctgatcaacg a 21 217 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 217 tcgttgatca
satctgtctg t 21 218 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 218 acagacagat
stgatcaacg a 21 219 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 219 tcgttgatca
catctgtctg t 21 220 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 220 acagacagat
gtgatcaacg a 21 221 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 221 gattctctcc
gagaaaacat c 21 222 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 222 gatgttttct
cggagagaat c 21 223 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 223 gattctctcc
ragaaaacat c 21 224 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 224 gatgttttct
yggagagaat c 21 225 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 225 gattctctcc
aagaaaacat c 21 226 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 226 gatgttttct
tggagagaat c 21 227 22 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 227 agtctcacac
atgtgcactc ac 22 228 22 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 228 gtgagtgcac
atgtgtgaga ct 22 229 22 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 229 agtctcacac
atgtgcactc ac 22 230 22 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 230 gtgagtgcac
atgtgtgaga ct 22 231 20 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 231 agtctcacac
gtgcactcac 20 232 20 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 232 gtgagtgcac
gtgtgagact 20 233 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 233 catgtgcact
gacgtggccg g 21 234 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 234 ccggccacgt
cagtgcacat g 21 235 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 235 catgtgcact
sacgtggccg g 21 236 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 236 ccggccacgt
sagtgcacat g 21 237 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 237 catgtgcact
cacgtggccg g 21 238 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 238 ccggccacgt
gagtgcacat g 21 239 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 239 ggggctgggg
ctgggtgcgt g 21 240 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 240 cacgcaccca
gccccagccc c 21 241 27 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 241 ggggctgggg
ctggggctgg gtgcgtg 27 242 27 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 242 cacgcaccca
ggccccaccc cagcccc 27 243 27 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 243 ggggctgggg
ctggggctgg gtgcgtg 27 244 27 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 244 cacgcaccca
gccccagccc cagcccc 27 245 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 245 tgtctaatta
tagaaatgga t 21 246 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 246 atccatttct
ataattagac a 21 247 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 247 tgtctaatta
yagaaatgga t 21 248 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 248 atccatttct
rtaattagac a 21 249 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 249 tgtctaatta
cagaaatgga t 21 250 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 250 atccatttct
gtaattagac a 21 251 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 251 ctgggaagtc
gtccctgacc c 21 252 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 252 gggtcaggga
cgacttccca g 21 253 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 253 ctgggaagtc
rtccctgacc c 21 254 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 254 gggtcaggga
ygacttccca g 21 255 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 255 ctgggaagtc
atccctgacc c 21 256 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 256 gggtcaggga
tgacttccca g 21 257 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 257 ccatgtcagc
gtgacacagg t 21 258 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 258 acctgtgtca
cgctgacatg g 21 259 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 259 ccatgtcagc
rtgacacagg t 21 260 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 260 acctgtgtca
ygctgacatg g 21 261 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 261 ccatgtcagc
atgacacagg t 21 262 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 262 acctgtgtca
tgctgacatg g 21 263 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 263 ctggtttttt
tcttccggtc a 21 264 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 264 tgaccggaag
aaaaaaacca g 21 265 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 265 ctggtttttt
tcttccggtc a 21 266 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 266 tgaccggaag
aaaaaaacca g 21 267 20 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 267 ctggtttttt
cttccggtca 20 268 20 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 268 tgaccggaag
aaaaaaccag 20 269 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 269 cactggcaca
gtggcctcta g 21 270 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 270 ctagaggcca
ctgtgccagt g 21 271 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 271 cactggcaca
rtggcctcta g 21 272 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 272 ctagaggcca
ytgtgccagt g 21 273 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 273 cactggcaca
atggcctcta g 21 274 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 274 ctagaggcca
ttgtgccagt g 21 275 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 275 cccaaaacac
gcacaccctg c 21 276 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 276 gcagggtgtg
cgtgttttgg g 21 277 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 277 cccaaaacac
rcacaccctg c 21 278 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 278 gcagggtgtg
ygtgttttgg g 21 279 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 279 cccaaaacac
acacaccctg c 21 280 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 280 gcagggtgtg
tgtgttttgg g 21 281 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 281 gtgcactcac
gtggccgggt g 21 282 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 282 cacccggcca
cgtgagtgca c 21 283 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 283 gtgcactcac
rtggccgggt g 21 284 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 284 cacccggcca
ygtgagtgca c 21 285 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 285 gtgcactcac
atggccgggt g 21 286 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 286 cacccggcca
tgtgagtgca c 21 287 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 287 ccacggcagc
cgtggacctg g 21 288 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 288 ccaggtccac
ggctgccgtg g 21 289 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 289 ccacggcagc
ygtggacctg g 21 290 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 290 ccaggtccac
rgctgccgtg g 21 291 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 291 ccacggcagc
tgtggacctg g 21 292 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 292 ccaggtccac
agctgccgtg g 21 293 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 293 ctccttccct
cgctaggtcc t 21 294 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 294 aggacctagc
gagggaagga g 21 295 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 295 ctccttccct
ygctaggtcc t 21 296 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 296 aggacctagc
ragggaagga g 21 297 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 297 ctccttccct
tgctaggtcc t 21 298 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 298 aggacctagc
aagggaagga g 21 299 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 299 gggaatcact
caacctctct g 21 300 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 300 cagagaggtt
gagtgattcc c 21 301 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 301 gggaatcact
maacctctct g 21 302 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 302 cagagaggtt
kagtgattcc c 21 303 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 303 gggaatcact
aaacctctct g 21 304 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 304 cagagaggtt
tagtgattcc c 21 305 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 305 tgtctccttt
cgcttctccc a 21 306 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 306 tgggagaagc
gaaaggagac a 21 307 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 307 tgtctccttt
ygcttctccc a 21 308 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 308 tgggagaagc
raaaggagac a 21 309 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 309 tgtctccttt
tgcttctccc a 21 310 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 310 tgggagaagc
aaaaggagac a 21 311 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 311 ccttaaacag
gatttgaaaa g 21 312 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 312 cttttcaaat
cctgtttaag g 21 313 21 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide 313 ccttaaacag satttgaaaa g 21 314 21 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide 314 cttttcaaat sctgtttaag g 21 315 21 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide 315 ccttaaacag catttgaaaa g 21 316 21 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide 316 cttttcaaat gctgtttaag g 21 317 21 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide 317 tgtgaccaca gatgagtgtg t 21 318 21 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide 318 acacactcat ctgtggtcac a 21 319 21 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide 319 tgtgaccaca ratgagtgtg t 21 320 21 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide 320 acacactcat ytgtggtcac a 21 321 21 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide 321 tgtgaccaca aatgagtgtg t 21 322 21 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide 322 acacactcat ttgtggtcac a 21 323 21 DNA
Artificial Sequence Description of Artificial Seq uence sequence of
G57998T (exon 10, Arg433Ser) 323 ac gct cag agt ttc atg gac t 21
Ala Gln Ser Phe Met Asp 1 5 324 6 PRT Artificial Sequence
Description of Artificial Sequence sequence of G57998T (exon 10,
Arg433Ser) 324 Ala Gln Ser Phe Met Asp 1 5 325 21 DNA Artificial
Sequence Description of Artificial Sequence sequence of G27258A
(exon 17, Arg723Gln) 325 gat tct ctc caa gaa aac atc 21 Asp Ser Leu
Gln Glu Asn Ile 1 5 326 7 PRT Artificial Sequence Description of
Artificial Sequence sequence of G27258A (exon 17, Arg723Gln) 326
Asp Ser Leu Gln Glu Asn Ile 1 5 327 21 DNA Artificial Sequence
Description of Artificial Sequence sequence of T249G (exon 8,
Phe329Cys) 327 ctc atg agc tgc ttc ttc aag 21 Leu Met Ser Cys Phe
Phe Lys 1 5 328 7 PRT Artificial Sequence Description of Artificial
Sequence sequence of T249G (exon 8, Phe329Cys) 328 Leu Met Ser Cys
Phe Phe Lys 1 5 329 20 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 329 taaccaggtt
gttgatcctc 20 330 20 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 330 gaggatcaac
aacctggtta 20 331 20 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 331 taaccaggtt
rttgatcctc 20 332 20 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 332 gaggatcaay
aacctggtta 20 333 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 333 tggggtgggg
atggcgcggg g 21 334 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 334 ccccgcgcca
tccccacccc a 21 335 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 335 tggggtgggg
rtggcgcggg g 21 336 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 336 ccccgcgcca
yccccacccc a 21 337 22 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 337 tgggcacgcg
accccccacg ca 22 338 22 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 338 tgcgtggggg
gtcgcgtgcc ca 22 339 22 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 339 tgggcacgcg
rccccccacg ca 22 340 22 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 340 tgcgtggggg
gycgcgtgcc ca 22 341 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 341 ccatgtgtcc
acgctggctt c 21 342 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 342 gaagccagcg
tggacacatg g 21 343 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 343 ccatgtgtcc
rcgctggctt c 21 344 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 344 gaagccagcg
yggacacatg g 21 345 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 345 tgaagccccc
aaccttgtgg g 21 346 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 346 cccacaaggt
tgggggcttc a 21 347 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 347 tgaagccccc
raccttgtgg g 21 348 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 348 cccacaaggt
ygggggcttc a 21 349 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 349 tgggtggcac
ggtgctggtg a 21 350 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 350 tcaccagcac
cgtgccaccc a 21 351 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 351 tgggtggcac
rgtgctggtg a 21 352 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 352 tcaccagcac
ygtgccaccc a 21 353 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 353 gctgggtggc
gcagtgctgg t 21 354 22 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 354 accaagcact
gcgccaccca gc 22 355 21 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 355 gctgggtggc
rcagtgctgg t 21 356 22 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 356 accaagcact
gygccaccca gc 22 357 20 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 357 ggcccgatca
cccgccgccg 20 358 20 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 358 cggcggcggg
tgatcgggcc 20 359 34 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 359 ggcccgatca
cccgccgccc ggtgcccgcc gccg 34 360 34 DNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide 360
cggcggcggg cagcgggcgg cgggtgatcg ggcc 34 361 56 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 361 tccctgcgcc gccgccgccg ccgccgccgc cgccgccgcc
gccgccagcg ctagcg 56 362 56 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 362 cgctagcgct
ggcggcggcg gcggcggcgg cggcggcggc ggcggcggcg caggga 56 363 59 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide 363 tccctgcgcc gccgccgccg ccgccgccgc cgccgccgcc
gccgccgcca gcgctagcg 59 364 59 DNA Artificial Sequence Description
of Artificial Sequence Synthetic oligonucleotide 364 cgctagcgct
ggcggcggcg gcggcggcgg cggcggcggc ggcggcggcg gcgcaggga 59 365 53 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide 365 tccctgcgcc gccgccgccg ccgccgccgc cgccgccgcc
gccagcgcta gcg 53 366 53 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 366 cgctagcgct
ggcggcggcg gcggcggcgg cggcggcggc ggcggcgcag gga 53 367 59 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide 367 tccctgcgcc gccgccgccg ccgccgccgc cgccgccgcc
gccgccgcca gcgctagcg 59 368 59 DNA Artificial Sequence Description
of Artificial Sequence Synthetic oligonucleotide 368 cgctagcgct
ggcggcggcg gcggcggcgg cggcggcggc ggcggcggcg gcgcaggga 59 369 50 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide 369 tccctgcgcc gccgccgccg ccgccgccgc cgccgccgcc
agcgctagcg 50 370 50 DNA Artificial Sequence Description of
Artificial Sequence Synthetic oligonucleotide 370 cgctagcgct
ggcggcggcg gcggcggcgg cggcggcggc ggcgcaggga 50 371 59 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide 371 tccctgcgcc gccgccgccg ccgccgccgc cgccgccgcc
gccgccgcca gcgctagcg 59 372 59 DNA Artificial Sequence Description
of Artificial Sequence Synthetic oligonucleotide 372 cgctagcgct
ggcggcggcg gcggcggcgg cggcggcggc ggcggcggcg gcgcaggga 59 373 47 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide 373 tccctgcgcc gccgccgccg ccgccgccgc cgccgccagc
gctagcg 47 374 47 DNA Artificial Sequence Description of Artificial
Sequence Synthetic oligonucleotide 374 cgctagcgct ggcggcggcg
gcggcggcgg cggcggcggc gcaggga 47 375 59 DNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide 375
tccctgcgcc gccgccgccg ccgccgccgc cgccgccgcc gccgccgcca gcgctagcg 59
376 59 DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide 376 cgctagcgct ggcggcggcg gcggcggcgg
cggcggcggc ggcggcggcg gcgcaggga 59 377 38 DNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide 377
tccctgcgcc gccgccgccg ccgccgccag cgctagcg 38 378 38 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 378 cgctagcgct ggcggcggcg gcggcggcgg cgcaggga 38
379 59 DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide 379 tccctgcgcc gccgccgccg ccgccgccgc
cgccgccgcc gccgccgcca gcgctagcg 59 380 59 DNA Artificial Sequence
Description of Artificial Sequence Synthetic oligonucleotide 380
cgctagcgct ggcggcggcg gcggcggcgg cggcggcggc ggcggcggcg gcgcaggga 59
381 20 DNA Artificial Sequence Description of Artificial Sequence
Synthetic oligonucleotide 381 tcaagcagag agagagtgtt 20 382 20 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide 382 aacactctct ctctgcttga 20 383 22 DNA Artificial
Sequence Description of Artificial Sequence Synthetic
oligonucleotide 383 tcaagcagag aaagagagtg tt 22 384 22 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide 384 aacactctct ttctctgctt ga 22 385 21 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide 385 ctggggcctt tgtgtcattc a 21 386 21 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide 386 tgaatgacac aaaggcccca g 21 387 21 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide 387 ctggggcctt ygtgtcattc a 21 388 21 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide 388 tgaatgacac raaggcccca g 21 389 21 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide 389 acacaaggag atgaagccgt t 21 390 21 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide 390 aacggcttca tctccttgtg t 21 391 21 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide 391 acacaaggag rtgaagccgt t 21 392 21 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide 392 aacggcttca yctccttgtg t 21 393 21 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide 393 caggcccccc cagacctcag g 21 394 21 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide 394 cctgaggtct gggggggcct g 21 395 21 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide 395 caggcccccc cagacctcag g 21 396 21 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide 396 cctgaggtct gggggggcct g 21 397 21 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide 397 tacagttttg attttgttga g 21 398 21 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide 398 ctcaacaaaa tcaaaactgt a 21 399 21 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide 399 tacagttttg rttttgttga g 21 400 21 DNA
Artificial Sequence Description of Artificial Sequence Synthetic
oligonucleotide 400 ctcaacaaaa ycaaaactgt a 21 401 11 PRT
Artificial Sequence Decsription of Artificial Sequence Illustrative
peptide 401 Thr Pro Leu Asn Lys Ile Lys Thr Ala Leu Gly 1 5 10 402
11 PRT Artificial Sequence Decsription of Artificial Sequence
Illustrative peptide 402 Thr Pro Leu Asn Lys Xaa Lys Thr Ala Leu
Gly 1 5 10 403 11 PRT Artificial Sequence Decsription of Artificial
Sequence Illustrative peptide 403 Cys Asn His Val Ser Thr Leu Ala
Ser Asn Tyr 1 5 10 404 11 PRT Artificial Sequence Decsription of
Artificial Sequence Illustrative peptide 404 Cys Asn His Val Ser
Xaa Leu Ala Ser Asn Tyr 1 5 10 405 15 DNA Artificial Sequence
Decsription of Artificial Sequence Synthetic oligonucleotide 405
cccgccgccc gggtg 15 406 14 PRT Artificial Sequence Description of
Artificial Sequence Illustrative transport family signature motif
406 Leu Ser Ser Gly Gly Gln Xaa Xaa Xaa Arg Xaa Xaa Xaa Ala 1 5
10
* * * * *