U.S. patent application number 10/831552 was filed with the patent office on 2005-01-27 for analysis and use of par1 polymorphisms for evaluating the risk of cardiovascular disorders.
This patent application is currently assigned to Aventis Pharma Deutschland GmbH. Invention is credited to Czech, Joerg, Deleuze, Jean-Francois, Kozian, Detlef, Mace, Sandrine, Ricard, Sylvain, Siegler, Karl-Ernst.
Application Number | 20050019795 10/831552 |
Document ID | / |
Family ID | 42667307 |
Filed Date | 2005-01-27 |
United States Patent
Application |
20050019795 |
Kind Code |
A1 |
Kozian, Detlef ; et
al. |
January 27, 2005 |
Analysis and use of PAR1 polymorphisms for evaluating the risk of
cardiovascular disorders
Abstract
The invention relates to polynucleotide sequences comprising
genetic variations of the PAR1 gene at positions 3090 and/or 3329.
The occurrence of these variants in humans correlates with
increased occurrence of particular cardiovascular disorders. The
invention furthermore relates to methods for detecting said genetic
variations for the purpose of patient diagnosis.
Inventors: |
Kozian, Detlef;
(Hattersheim, DE) ; Czech, Joerg; (Marburg,
DE) ; Siegler, Karl-Ernst; (Ludwigshafen, DE)
; Deleuze, Jean-Francois; (Combs La Ville, FR) ;
Ricard, Sylvain; (Paris, FR) ; Mace, Sandrine;
(Jouy-En-Josas, FR) |
Correspondence
Address: |
ROSS J. OEHLER
AVENTIS PHARMACEUTICALS INC.
ROUTE 202-206
MAIL CODE: D303A
BRIDGEWATER
NJ
08807
US
|
Assignee: |
Aventis Pharma Deutschland
GmbH
Frankfurt
DE
|
Family ID: |
42667307 |
Appl. No.: |
10/831552 |
Filed: |
April 23, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60503732 |
Sep 17, 2003 |
|
|
|
60465337 |
Apr 25, 2003 |
|
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Current U.S.
Class: |
435/6.11 ;
536/23.2 |
Current CPC
Class: |
C07H 17/08 20130101;
C12Q 1/6883 20130101; C12Q 2600/156 20130101; C07K 14/723
20130101 |
Class at
Publication: |
435/006 ;
536/023.2 |
International
Class: |
C12Q 001/68; C07H
021/04 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 24, 2003 |
DE |
10318496.1 |
Claims
What is claimed is:
1. An isolated polynucleotide sequence which is a polymorphism of a
PAR1 gene, wherein the isolated polynucleotide sequence comprises
SEQ ID NO: 4.
2. An isolated polynucleotide sequence which is a polymorphism of
the PAR1 gene, wherein the isolated polynucleotide sequence
comprises SEQ ID NO: 8.
3. An isolated polynucleotide sequence which is a polymorphism of
the PAR1 gene, wherein the polynucleotide sequence comprises that
portion of SEQ ID NO: 4 from nucleotide 3090 to nucleotide
3329.
4. A method of determining a patient's relative risk of a
cardiovascular disorder, comprising a. obtaining a nucleic acid
sample from the patient; and b. determining the presence in the
nucleic acid sample of at least one single nucleotide polymorphism
of the PAR1 gene sequence, selected from the group of polymorphisms
corresponding to a C for T substitution at position 3090 and a C
for A substitution at position 3329 of SEQ ID NO: 1; wherein the
presence of the at least one polymorphism is diagnostic of a
patient's relative risk of atrial fibrillation, acute coronary
syndrome, cardiomyopathy or unstable angina.
5. The method of claim 4, wherein the step of determining the
presence of at least one single nucleotide polymorphism comprises
amplification of a portion of the nucleic acid sample comprising
position 3090 of SEQ ID NO: 1 by polymerase chain reaction.
6. The method of claim 4, wherein the step of determining the
presence of at least one single nucleotide polymorphism comprises
amplification of a portion of the nucleic acid sample comprising
position 3329 of SEQ ID NO: 1 by polymerase chain reaction.
7. The method of claim 4, wherein the step of determining the
presence of at least one single nucleotide polymorphism comprises
sequencing of a portion of the nucleic acid sample comprising
position 3090 of SEQ ID NO: 1.
8. The method of claim 4, wherein the step of determining the
presence of at least one single nucleotide polymorphism comprises
sequencing of a portion of the nucleic acid sample comprising
position 3329 of SEQ ID NO: 1.
Description
FIELD OF THE INVENTION
[0001] The invention relates to polynucleotide sequences comprising
genetic variations of the PAR1 gene at positions 3090 and/or 3329,
and their use in determining a patient's risk of cardiovascular
disorders.
BACKGROUND OF THE INVENTION
[0002] The protease-activated receptor 1 (PAR1) is a thrombin
receptor which belongs to the class of G protein-coupled receptors
(GCPR). The gene for PAR1 is located on chromosome 5q13, consists
of two exons and covers a region of approx. 27 kb. PAR1 is
expressed in, inter alia, endothelial cells, smooth muscles cells,
fibroblasts, neurons and human platelets. In platelets, PAR1 is an
important signal transduction receptor which is involved in the
initiation of platelet aggregation.
[0003] PARs are activated via proteolytic removal of a part of the
N terminus of said PARs, whereby a new N-terminal sequence is
exposed which then activates the receptor.
[0004] PAR1 and PAR4 play a central part in the activation of
platelets; the activation of these receptors in platelets leads to
morphological changes, release of ADP and aggregation of said
platelets.
[0005] A connection of coronary heart diseases with single
nucleotide polymorphisms (SNP) in the promoter region of PAR1 in a
group of Korean patients was not confirmed. In another study, a
PAR1 promoter variant was shown to have a protective action for the
development of venous thromboembolisms.
[0006] The sequence of the human PAR1 gene is known. The
polynucleotide sequence of this gene can be accessed under the
number NM-001992 at the NCBI nucleotide database. Likewise, the
protein sequence is available under the number NP-001983 at the
NCBI protein database. NCBI is the National Center for
Biotechnology Information (postal address: National Center for
Biotechnology Information, National Library of Medicine, Building
38A, Bethesda, Md. 20894, USA; Web address: www.ncbi.nhm.nih.gov).
The cloning of the PAR1 gene has been described, inter alia, in
"Schmidt et al., J. Biol. Chem. 271, 9307-9312, 1996".
DESCRIPTION OF THE INVENTION
[0007] There are various new polymorphisms of the PAR1 gene, by
means of which it is possible to determine a relatively strong
disposition of an individual for coronary heart diseases. The
affected individuals are thus enabled to counteract this risk
factor in time by adapting their life style accordingly, for
example by compensating via increased control of other damaging
influences such as smoking, alcohol consumption, cholesterol-rich
food, high blood pressure etc.
[0008] Such health-related preventive mechanisms would not be
possible without knowledge of the PAR1 polymorphisms which are
explained in more detail below and the use thereof in corresponding
methods.
[0009] Variants of a particular nucleotide sequence with
substitutions at individual positions are known to the skilled
worker under the term SNP (=single nucleotide polymorphism).
[0010] The invention relates to an isolated polynucleotide sequence
of the PAR1 gene, which comprises a C for T substitution at
position 3090 of the PAR1 sequence according to NM-001992 which, as
prior art, is publicly available. In a preferred embodiment, the
polynucleotide sequence of the PAR1 gene having a T to C
substitution at position 3090 encompasses a sequence according to
SEQ ID NO: 2 and, in a particularly preferred embodiment of said
polynucleotide sequence, the latter comprises a sequence of SEQ ID
NO: 2.
[0011] The invention furthermore relates to an isolated
polynucleotide sequence of the PAR1 gene, which comprises an C for
A substitution at position 3329 of the PAR1 sequence according to
NM-001992 which, as prior art, is publicly available. In a
preferred embodiment, the polynucleotide sequence of the PAR1 gene
having an A to C substitution at position 3329 encompasses a
sequence according to SEQ ID NO: 3 and, in a particularly preferred
embodiment of said polynucleotide sequence, the latter comprises a
sequence of SEQ ID NO: 3.
[0012] The invention also relates to an isolated polynucleotide
sequence of the PAR1 gene, which comprises a C for T substitution
at position 3090 of the PAR1 sequence according to NM-001992 and,
simultaneously, a V for A substitution at position 3329 of said
PAR1 sequence. In a preferred embodiment, the polynucleotide
sequence of the PAR1 gene having a T to C substitution at position
3090 and a simultaneous A to C substitution at position 3329
encompasses a sequence according to SEQ ID NO: 4 and, in a
particularly preferred embodiment of said polynucleotide sequence,
the latter comprises a sequence of SEQ ID NO; 4.
[0013] The invention also relates to an isolated part of the
polynucleotide sequence of the PAR1 gene, which comprises a
sequence according to SEQ ID NO: 5.
[0014] The invention also relates to an isolated part of the
polynucleotide sequence of the PAR1 gene, which sequence comprises
a C for T substitution at position 3090, based on the PAR1 sequence
according to NM-001992, which part comprises a sequence according
to SED ID NO: 6.
[0015] The invention also relates to an isolated part of the
polynucleotide sequence of the PAR1 gene, which sequence comprises
a C for A substitution at position 3329, based on the PAR1 sequence
according to NM-001992, which part comprises a sequence according
to SED ID NO: 7.
[0016] The invention also relates to an isolated part of the
polynucleotide sequence of the PAR1 gene, which sequence comprises
a C for T substitution at position 3090, based on the PAR1 sequence
according to NM-001992, and simultaneously a C for A substitution
at position 3329 of said PAR1 sequence, which part comprises a
sequence according to SEQ ID NO: 8.
[0017] The invention furthermore comprises the preparation of a
3592 base pair polynucleotide sequence of the PAR1 cDNA gene, which
sequence may or may not comprise the polymorphisms at positions
3090 and 3329, as defined above, individually or in combination,
which preparation comprises the following method steps:
[0018] a] Providing human cDNA comprising a PAR1 sequence according
to SEQ ID NO: 2 and/or a PAR1 sequence according to SEQ ID NO: 3
and/or a PAR1 sequence according to SEQ ID NO: 4,
[0019] b] Providing a primer pair having a sequence according to
SEQ ID NO:
[0020] 9 and SEQ ID NO: 10.
[0021] c] Amplifying the PAR1 polynucleotide sequence by the
polymerase chain extension reaction (PCR),
[0022] d] Isolating and/or purifying the 3.56 kb fragment obtained
from c],
[0023] e] Sequencing the fragment from d].
[0024] The invention also relates to the preparation of a
polynucleotide sequence according to SEQ ID NO: 5, SEQ ID NO: 6,
SEQ ID NO: 7 or SEQ ID NO: 8, which preparation comprises the
following method steps:
[0025] a] Providing human genomic DNA comprising a PAR1 sequence
according to SEQ ID NO: 1 and/or a PAR1 sequence according to SEQ
ID NO: 2 and/or a PAR1 sequence according to SEQ ID NO: 3 and/or a
PAR1 sequence according to SEQ ID NO: 4
[0026] b] Providing a primer pair according to SEQ ID NO: 11 and
SEQ ID NO: 12
[0027] c] Amplifying the fragment of the PAR1 polynucleotide
sequence by the polymerase chain extension reaction (PCR),
[0028] d] Isolating and/or purifying the fragment obtained from
c],
[0029] e] Sequencing the fragment from d].
[0030] The invention furthermore relates to a method for detecting
whether or not there is in a PAR1 gene a T to C substitution at
position 3090 of the sequence according to NM-001992 and/or an A to
C substitution at position 3329 of the sequence according to
NM-001992, which method comprises the following method steps:
[0031] a] Providing biological material comprising human cells,
[0032] b] Obtaining chromosomal DNA from the material of a],
[0033] c] Amplifying a polynucleotide fragment by means of the
primers according to SEQ ID NO: 11 and SEQ ID NO: 12, using a PCR
reaction,
[0034] d] Sequencing the polynucleotide fragment from c].
[0035] The invention furthermore relates to a method for detecting,
whether or not there is in a PAR1 gene a T to C substitution at
position 3090 of the sequence according to NM-001992 and/or an A to
C substitution at position 3329 of the sequence according to
NM-001992, which method comprises the following method steps:
[0036] a] Providing biological material comprising human cells,
[0037] b] Obtaining RNA from the material of a],
[0038] c] Transcribing said RNA to cDNA by means of reverse
transcriptase,
[0039] d] Possibly amplifying a polynucleotide fragment by means of
the primers according to SEQ ID NO: 10 and SEQ ID NO: 11, using
said PCR reaction,
[0040] e] Sequencing the cDNA from c] and/or the polynucleotide
fragment from d].
[0041] The invention also relates to a method for detecting whether
or not there is in a PAR1 gene a T to C substitution at position
3090 of the sequence according to NM-001992 and/or an A to C
substitution at position 3329 of the sequence according to
NM-001992, which method comprises the following method steps:
[0042] a] Providing biological material comprising human cells,
[0043] b] Obtaining chromosomal DNA from the material of a],
[0044] c] Southern blotting the chromosomal DNA from b],
[0045] d] Providing a probe according to SEQ ID NO: 5 and/or SEQ ID
NO: 6 and/or SEQ ID NO: 7 and/or SEQ ID NO: 8,
[0046] e] Hybridizing the Southern blot from c] with the probe form
d] under stringent hybridization conditions,
[0047] f] Determining the presence or absence of a genetic
variation in the PAR1 gene at position 3090 and/or 3329 according
to NM-001992 by comparing the results of the hybridization from
e].
[0048] The invention furthermore relates to a method for detecting
whether or not there is in a PAR1 gene a T to C substitution at
position 3090 of the sequence according to NM-001992 and/or an A to
C substitution at position 3329 of the sequence according to
NM-001992, which method comprises the following method steps:
[0049] a] Providing biological material comprising human cells,
[0050] b] Obtaining RNA from the material of a],
[0051] c] Northern blotting the RNA from b],
[0052] d] Providing a probe according to SEQ ID NO: 5 and/or SEQ ID
NO: 6 and/or SEQ ID NO: 7 and/or SEQ ID NO: 8,
[0053] e] Hybridizing the Northern blot form c] with the probe from
d] under stringent hybridization conditions,
[0054] f] Determining the presence or absence of a genetic
variation in the PAR1 gene at position 3090 and/or 3329 according
to NM-001992 by comparing the results of the hybridization.
[0055] Detection of the genetic variations or polymorphisms in the
PAR1 gene at positions 3090 and/or 3329 may be used as (a) genetic
marker for evaluating the risk of atrial fibrillation, acute
coronary syndrome, cardiomyopathy and/or unstable angina, as (b)
marker for preventive treatment for atrial fibrillation, acute
coronary syndrome, cardiomyopathy and/or stable angina of the
carriers of the corresponding genetic variants, as (c) marker for
adjusting the dose of a pharmaceutically active substance to be
administered for atrial fibrillation, acute coronary syndrome,
cardiomyopathy and/or unstable angina, as (d) marker for
determining the high throughput-screening strategy for identifying
a pharmaceutically active substance for atrial fibrillation, acute
coronary syndrome, cardiomyopathy and/or unstable angina, as (e)
marker for identifying the relevant individuals or patients for
clinic studies in order to test the tolerability, safety and
efficacy of a pharmaceutical substance for atrial fibrillation,
acute coronary syndrome, cardiomyopathy and/or unstable angina, and
as (e) basis for developing assays systems for analyzing the
genetic variation in the PAR1 gene at the DNA, RNA or protein
level.
[0056] The invention also relates to an isolated polynucleotide
sequence having from 21 to 50 nucleotides, which comprises a
sequence according to SEQ ID NO: 11. Said sequence preferably
comprises SEQ ID NO: 11. The invention furthermore relates to an
isolated polynucleotide sequence having from 20 to 50 nucleotides,
which comprises a sequence according to SEQ ID NO: 12. Said
sequence preferably comprises SEQ ID NO: 12.
[0057] The invention also relates to the use of an isolated
polynucleotide sequence having from 21 to 50 nucleotides, which
encompasses or comprises a sequence according to SEQ ID NO: 11, in
combination with an isolated polynucleotide sequence having from 20
to 50 nucleotides, which encompasses or comprises a sequence
according to SEQ ID NO: 12, for amplifying a corresponding fragment
of the PART1 gene by means of the polymerase chain extension
reaction (PCR). This use preferably relates to the amplification of
a fragment of a PAR1 gene having a T to C substitution at position
3090 of the sequence according to NM-001992 and/or having an A to C
substitution at position 3329 of the sequence according to
NM-001992.
[0058] Moreover, the invention comprises a kit of parts which
comprises
[0059] a] an isolated polynucleotide sequence of from 21 to 50
nucleotides in length, which encompasses or comprises a sequence
according to SEQ ID NO: 11,
[0060] b] an isolated polynucleotide sequence of from 20 to 50
nucleotides in length, which encompasses or comprises a sequence
according to SEQ ID NO: 12,
[0061] c] at least one enzyme for carrying out the polymerase chain
extension reaction (PCR),
[0062] d] possibly substances and/or solutions for carrying out the
polymerase chain extension reaction,
[0063] e] possibly polynucleotide sequences encompassing the PAR1
gene with or without substitution at position 3090 of the PAR1
sequence according to NM-001992 and/or position 3329 according to
NM-001992 in full length and/or parts thereof
[0064] f] and possibly reagents for carrying out the sequencing
reaction.
[0065] Kit of parts here and below means the combination of said
components which have been combined into a functional unit in
spatial juxtaposition to each other.
[0066] The invention furthermore relates to the preparation of the
above-described kit of parts, which comprises
[0067] a] providing an isolated polynucleotide sequence of from 21
to 50 nucleotides in length, which encompasses or comprises a
sequence according to SEQ ID NO: 11,
[0068] b] providing an isolated polynucleotide sequence of from 20
to 50 nucleotides in length, which encompasses or comprises a
sequence according to SEQ ID NO: 12,
[0069] c] providing an enzyme for carrying out the polymerase chain
extension reaction (PCR),
[0070] d] providing, where appropriate, reagents for carrying out a
sequencing
[0071] e] possibly providing substances and/or solutions for
carrying out said polymerase chain extension reaction (PCR)
[0072] f] possibly providing polynucleotide sequences comprising
the PAR1 gene with or without a T to C substitution at position
3090 of the PAR1 sequence according to NM-001992 and/or an A to C
substitution at position 3329 according to NM-001992, in each case
in the full length, or parts thereof,
[0073] g] introducing the components from a] to f] in each case
separately into suitable containers,
[0074] h] combining, where appropriate, the containers from g] in
one or more pack units.
[0075] The above-described kit of parts may be used for amplifying
a fragment of the PAR1 gene.
BRIEF DESCRIPTION OF THE FIGURES
[0076] FIG. 1 depicts the characteristics of the study group.
[0077] FIG. 2 depicts the distribution of PAR1 variants T3090C and
A3329C in 1362 individuals analyzed.
[0078] FIG. 3 depicts the association of PAR1 variants C3090C with
atrial fibrillation and cardiomyopathy.
[0079] FIG. 4 depicts the association of PAR1 variants C3329C with
atrial fibrillation, acute coronary syndrome and unstable
angina.
[0080] FIG. 5 depicts the polynucleotide sequence of the cDNA of
the human PAR1 gene in 5'/3' orientation. The sequence corresponds
to the sequence made publicly available by the NCBI Nucleotide
Database under number NM-001992. The prepared sequence is identical
to SEQ ID NO: 1.
[0081] FIG. 6 depicts the polynucleotide sequence of the cDNA of
the human PAR1 gene in 5'/3' orientation with a polymorphism at
position 3090 of the sequence according to NM-001992, which
polymorphism comprises a T to C substitution. The depicted sequence
is identical to SEQ ID NO: 2.
[0082] FIG. 7 depicts the polynucleotide sequence of the cDNA of
the human PAR1 gene in 5'/3' orientation with a polymorphism at
position 3329 of the sequence according to NM-001992, which
polymorphism comprises an A to C substitution. The depicted
sequence is identical to SEQ ID NO: 3.
[0083] FIG. 8 depicts the polynucleotide sequence of the cDNA of
the human PAR1 gene in 5'/3' orientation with a polymorphism at
position 3090 of the sequence according to NM-001992, which
polymorphism comprises a T to C substitution, and with a
simultaneous second polymorphism at position 3329 of the sequence
according to NM-001992, which polymorphism comprises an A to C
substitution. The depicted sequence is identical to SEQ ID NO:
4.
[0084] FIG. 9 depicts the polynucleotide sequence of a fragment of
the human PAR1 gene in 5'/3' orientation. The depicted sequence is
identical to SEQ ID NO: 5.
[0085] FIG. 10 depicts the polynucleotide sequence of a fragment of
the human PAR1 gene in 5'/3' orientation with a polymorphism at
position 3090 of the sequence according to NM-01992, which
polymorphism comprises a T to C substitution. The depicted sequence
is identical to SEQ ID NO: 6.
[0086] FIG. 11 depicts the polynucleotide sequence of a fragment of
human PAR1 gene in 5'/3' orientation with a polymorphism at
position 3329 of the sequence according to NM-001992, which
polymorphism comprises an A to C substitution. The depicted
sequence is identical to SEQ ID NO: 7.
[0087] FIG. 12 depicts the polynucleotide sequence of a fragment of
the human PAR1 gene in 5'/3' orientation with a polymorphism at
position 3090 of the sequence according to NM-001992, which
polymorphism comprises a T to C substitution, and with a
simultaneous second polymorphism at position 3329 of the sequence
according to NM-001992, which polymorphism comprises an A to C
substitution. The depicted sequence is identical to SEQ ID NO:
8.
[0088] FIG. 13 depicts the polynucleotide sequence in 5'/3'
orientation of the 5' end of the cDNA of the human PAR1 gene. The
depicted sequence is identical to SEQ ID NO: 9.
[0089] FIG. 14 depicts the polynucleotide sequence in 5'/3'
orientation of the 3' end of the cDNA of the human PAR1 gene. The
depicted sequence is identical to SEQ ID NO: 10.
[0090] FIG. 15 depicts the polynucleotide sequence in 5'/3'
orientation of the cDNA of the human PAR1 gene, relating to
positions 2767 to 2789 according to NM-001992. The depicted
sequence is identical to SEQ ID NO: 11.
[0091] FIG. 16 depicts the polynucleotide sequence in 5'/3'
orientation of the Exon No. 1 of the cDNA of the human PAR1 gene.
The depicted sequence is identical to SEQ ID NO: 12.
[0092] FIG. 17 depicts the protein sequence of the human PAR1
receptor. The sequence corresponds to the sequence made publicly
available by the NCBI Protein Database under number NP-001983. The
depicted sequence is identical to SEQ ID NO: 13.
EXAMPLES
[0093] The technical aspects of the invention are discussed in more
detail in the following embodiments.
[0094] Isolated polynucleotide sequences of the PAR1 gene may be
prepared, for example, by amplification by means of the polymerase
chain extension reaction (PCR). Suitable primers for this purpose
are described in SEQ ID NO: 9 and SEQ ID NO: 10.
[0095] The PCR is an in-vitro technique which may be used to
selectively duplicate polynucleotide sections which are flanked by
two known sequences. Amplification requires short, single-stranded
DNA molecules which are complementary to the ends of a defined
sequence of a DNA or RNA template (primers). A DNA polymerase
extends the primers, under the correct reaction conditions and in
the presence of deoxynucleotide triphosphates (dNTPs), along the
single-stranded and denatured polynucleotide template and thus
synthesizes new DNA strands whose sequence is complementary to said
template. During this process, the temperature is changed at
regular intervals so that, time after time, the polynucleotide
strands are denatured and the primers can be attached and extended.
Heat-stable DNA polymerases, for example Taq polymerase, are used.
A typical PCR reaction mixture contains, apart from a
polynucleotide template, two suitable primer nucleotides, for
example at concentrations between 0.2 to 2 .mu.M, furthermore
dNTPs, for example at concentrations of 200 .mu.M per dNPT,
furthermore MgCl.sub.2 having a concentration of 1-2 mM, and 1-10
units of a heat-stable DNA polymerase such as, for example, Taq
polymerase (Thermus aquaticus polymerase). Heat-stable DNA
polymerase and the components for carrying out the same, and also
protocols, are commercially supplied by numerous companies such as,
for example, Roche Diagnostics, Clontech, Life Technologies, New
England Biolabs, Promega, Stratagene, etc.
[0096] The polynucleotide template for amplifying the
polynucleotide sequence to be isolated may be present in the form
of RNA or DNA. If the polynucleotide template is RNA, then the
latter is transcribed to DNA by means of reverse transcriptase,
prior to the actual PCR reaction. The amount of polynucleotide
template for carrying out the PCR reaction may be from 0.01 to 20
ng, for example.
[0097] The polynucleotide template is obtained using techniques
known to the skilled worker for obtaining DNA and/or RNA from
biological material. Biological material should include here, inter
alia, the cells of a tissue or organ (e.g. brain, blood, liver,
spleen, kidney, heart, blood vessels) of a vertebrate, including
humans, or cells from a eukaryotic cell culture (e.g. Hela cells,
CHO cells, 3T3 cells) or cells comprising bacteria or yeasts in
which the DNA sequence to be isolated is present in cloned
form.
[0098] Cells of a tissue assemblage or organ of a vertebrate,
including humans, may be obtained by taking blood, tissue puncture
or surgical techniques. A polynucleotide template may be obtained
therefrom, for example, by disrupting the cells, possibly
concentrating individual organelles, in particular the nucleus, and
recovering the DNA or RNA by precipitation and centrifugation.
[0099] Another method for preparing isolated polynucleotide
sequences of the PAR1 gene comprises cloning the PAR1 gene,
subsequently expressing it in bacteria or yeast and purifying the
expressed polynucleotide. The previously mentioned PCR reaction,
for example, is suitable for preparing a polynucleotide fragment
which is clonable. It is advantageous to use, for a fragment to be
cloned, primers which carry the recognition sequence of a reaction
enzyme 5' of the complementary sequence. The two primers may use in
each case the same or different recognition sequences for
restriction enzymes.
[0100] Examples of common restriction enzymes are: BamHI (GGATCC),
ClaI (ATCGAT), EcOR1 (GMTTC), EcORV (GATATC), HindIII (AAGCTT) NcoI
(CCATGG) Sail (GTCGAC), XbaI (TCTAG1).
[0101] For cloning, a vector is treated with the restriction
enzymes which correspond to the recognition sequences attached to
the primers. The fragment is connected to the vector by means of
ligase by isolation and treatment with the same restriction
enzymes. Vector means a DNA molecule such as, for example, a
plasmid, bacteriophage or a cosmid, with the aid of which it is
possible to clone genes or other DNA sequences and to introduce
them into a bacterial or eukaryotic cell for replication. Examples
of vectors are DNA molecules such as pBR322, pUC18/19, pBluescript,
pcDNA3.1. Vectors are commercially available from specialist
companies for biotechnological material, such as Roche Diagnostics,
New England Biolabs, Promega, Stratagene etc.
[0102] The instructions required for carrying out the PCR reaction,
for providing polynucleotides or for carrying out cloning
procedures can be found by the skilled worker in the form of
recipes and protocols in standard manuals such as, for example, in
a] "Current Protocols in Molecular Biology by Frederick M. Ausubel
(Editor), Roger Brent (Editor), Robert E. Kingston (Editor), David
D. Moore (Editor), J. G. Seidman (Editor), Kevin Struhl (Editor),
loose leaf edition, continuously updated, John Wiley & Sons,
Inc., New York or in b] Short Protocols in Molecular Biology, 5th
edition, by Frederick M. Ausubel (Editor), Roger Brent (Editor),
Robert E. Kingston (Editor), David D. Moore (Editor), J. G. Seidman
(Editor), John A. Smith (Editor), Kevin Struhl (Editor), October
2002, John Wiley & Sons, Inc., New York" or in c] "Molecular
Cloning by J. Sambrock, E. F. Fritsch, T. Maniatis; Cold Spring
Harbor Laboratory Press".
[0103] Suitable primer sequences are provided, for example, via
chemical synthesis thereof which may be carried out commercially to
order by companies such as MWG Biotech, etc.
[0104] Human cDNA from different organs is commercially available
from companies such as, for example, Promega, Stratagene or
others.
[0105] The sequencing of a polynucleotide is carried out by means
of routine methods known to the skilled worker by using, for
example, laboratory robots from companies such as, for example,
Life Technologies, Applied Biosystems, BiORad or others.
[0106] Isolated polynucleotide sequences of the PAR1 variant and
fragments therefrom may also be used for hybridization at different
stringencies. Stringency describes reaction conditions which
influence the specificity of hybridization or attachment of two
single-stranded nucleic acid molecules. The stringency and thus
also specificity of a reaction can be increased by increasing the
temperature and lowering the ionic strength. Low stringency
conditions are present, for example, if the hybridization is
carried out at room temperature in 2.times.SSC solution. High
stringency conditions are present, for example, if hybridization is
carried out at 68.degree. C. in 0.1.times.SSC/0.1% SDS
solution.
[0107] Hybridization under stringent hybridization conditions in
accordance with the present application means:
[0108] 1] Hybridizing the labeled probe with the sample to be
studied at 65.degree. C. (or, in the case of oligonucleotides,
5.degree. C. below the melting temperature) overnight in 50 mM Tris
pH 7.5, 1 NaCl, 1% SDS, 10% dextran sulfate, 0.5 mg/ml denatured
salmon sperm DNA.
[0109] 2] Washing at room temperature in 2.times.SSC for 10
min.
[0110] 3] Washing at 65.degree. C. (or, in the case of
oligonucleotides, 5.degree. C. below the melting temperature) in
1.times.SSC/1% SDS for 30 min.
[0111] 4] Washing at 65.degree. C. (or, in the case of
oligonucleotides, 5.degree. C. below the melting temperature) in
0.2.times.SSC/0.1% SDS for 30 min.
[0112] 5] Washing at 65.degree. C. (or, in the case of
oligonucleotides, 5.degree. C. below the melting temperature) in
0.1% SSC/0.1% SDS for 30 min.
[0113] DNA fragments of 20 nucleotides in overall length are to be
regarded as being oligonucleotides for this purpose. The melting
temperature results from the formula T.sub.m=2 (number of A+T)+4
(number of G+C)C.degree..
[0114] A 2.times.SSC or 0.1.times.SSC solution is prepared by
diluting a 20.times.SSC solution accordingly. The 20.times.SSC
solution comprises a 3M NaCl/0.3 sodium citrate 2H.sub.2O solution.
SDS is sodium dodecyl sulfate.
[0115] The hybridization is carried out by transferring the
polynucleotides to be studied to a nylon or nitrocellulose membrane
(Southern blot--DNA; Northern blot--RNA), after electrophoretic
fractionation and subsequent denaturation. The hybridization is
carried out using a probe which is radio-labeled or has been
labeled in another way, for example with the aid of fluorescent
dyes. The probe comprises a usually single-stranded and/or
denatured DNA or RNA polynucleotide sequence which binds to the
complementary nucleotide sequence of the once again single-stranded
and/or denatured DNA or RNA polynucleotide sequence to be
studied.
[0116] Single nucleotide polymorphisms of the PAR1 gene may be
detected with the aid of the primers of the invention, also by SSCP
analysis. SSCP stands for Single Stranded Conformation Polymorphism
which is an electrophoretic technique for identifying individual
base pair substitutions. The polynucleotides to be studied are
amplified by PCR by means of labeled primers and, after
denaturation into single strands, fractionated in a polyacrylamide
gel electrophoresis (PAGE). If the DNA fragments to be studied
exhibit individual base pair substitutions, they then possess
different conformations and thus migrate in the PAGE at different
rates.
[0117] Examples of substances for carrying out the PCR are buffers
such as Hepes or Tris, furthermore dAPP, dGTP, dTTP, dCTP, and
Mg.sup.2+ and possibly further divalent or monovalent irons.
Solutions contain these substances in dissolved form.
[0118] Amplification of Genomic Regions of the PAR1 Gene
[0119] The T to C nucleotide substitution at position 3090 and the
A to C substitution at position 3329 in the PAR1 sequence were
detected using the following primers:
1 (SEQ ID NO: 11) Primer 1: 5'-ACAGACTGGAATAAGACAGAG-3' (SEQ ID NO:
12) Primer 2: 5'-CCAGTGCTAGCTTCTACTTA- C-3
[0120] Primer 1 (SEQ ID NO: 11) corresponds to positions 2767 to
2789 of the NM-001992 reference sequence. Primer 2 is derived from
Exon No. 1 of the PAR1 gene.
[0121] PCR Protocol for the Amplification:
[0122] The reagents used are from Applied Biosystems (Foster City,
USA): 20 ng of genomic DNA; 1 unit of TaqGold DNA polymerase;
1.times. Taq polymerase buffer; 500 .mu.M of dNTPs; 2.5 mM
MgCl.sub.2: 200 nM of each amplification primer pair; H.sub.2O to 5
.mu.l.
[0123] PCR Amplification Program for the Genotyping
2 95.degree. C. for 10 min .times.1 cycle 95.degree. C. for 30 sec
.times.2 cycles 70.degree. C. for 30 sec 95.degree. C. for 30 sec
.times.2 cycles; 65.degree. C. for 30 sec 95.degree. C. for 30 sec
.times.2 cycles; 60.degree. C. for 30 95.degree. C. for 30 sec
.times.40 cycles; 56.degree. C. for 30 sec 72.degree. C. for 30 sec
72.degree. C. for 10 min .times.1 cycle; 4.degree. C. for 30
sec
[0124] Identification of SNPs
[0125] Protocol for the Minisequencing and Detection of the
SNPs.
[0126] All reagents are from Applied Biosystems (Foster City, USA).
2 .mu.l of purified PCR product, 1.5 .mu.L of BigDye Terminator
Kit, 200 nM sequencing primer; H.sub.20 to 10 .mu.l.
[0127] Amplification program for the sequencing:
3 96.degree. C. for 2 min .times.1 cycle 96.degree. C. for 10 sec
.times.30 cycles 55.degree. C. for 10 sec 65.degree. C. for 4 min
72.degree. C. for 7 min .times.1 cycle; 4.degree. C. for 30 sec
[0128] Analysis of the Sequencing Products:
[0129] The sequences were first analyzed using the sequence
analysis software (Applied Biosystems, Foster City, USA) to obtain
the raw data, then processed using Phred, Phrap, Polyphred and
Consed. Phred, Phrap, Polyphred and Consed are software written by
Phil Green at Washington University
(http://www,genome.washington.edu).
[0130] Assigning PAR1 SNPs to Coronary Disorders
[0131] In a clinical study, two PAR1 polymorphisms from the
3'-noncoding region of the gene were studied for a connection with
thrombotic and cardiovascular complications in a cohort of
patients.
[0132] The following abbreviations are used below (all positions
indicated refer to the nucleotide positions in the reference
sequence NM-001992).
[0133] PAR1 T3090T describes the group of individuals whose alleles
of the PAR1 gene both have a thymidine (T) at position 3090. These
individuals are homozygous with respect to this PAR1 variant.
[0134] PAR1 T3090C describes the group of individuals whose one
allele of the PAR1 gene has a cytidine (C) at position 3090 and
whose other allele of the PAR1 gene has a thymidine (T) at position
3090. These individuals are heterozygous with respect to this PAR1
variant.
[0135] PAR1 C3090C describes the group of individuals whose alleles
of the PAR1 gene both have a cytidine (C) at position 3090. These
individuals are homozygous with respect to this PAR1 variant.
[0136] PAR1 A3329A describes the group of individuals whose alleles
of the PAR1 gene both have an adenosine (A) at position 3329. These
individuals are homozygous with respect to this PAR1 variant.
[0137] PAR1 A3329C describes the group of individuals whose one
allele of the PAR1 gene has a cytidine (C) at position 3329 and
whose other allele of the PAR1 gene has an adenosine (A) at
position 3329. These individuals are heterozygous with respect to
this PAR1 variant.
[0138] PAR1 C3329C describes the group of individuals whose alleles
of the PAR1 gene both have a cytidine (C) at position 3329. These
individuals are homozygous with respect to this PAR1 variant.
[0139] In the group of patients analyzed (FIG. 1), statistically
significant associations of the homozygous carriers of the PAR1
variant C3090C with atrial fibrillation and cardiomyopathy were
observed. After carrying out a logistic regression, a 1.97 fold
increased risk of atrial fibrillation and a 1.84 fold increased
risk of cardiomyopathy were found in homozygous carriers of the
PAR1 variant C3090C compared to carriers of the PAR1 variants
T3090/T3090T (FIG. 3).
[0140] It was shown that, for carriers of the PAR1 variant C3329C,
said variant is associated with a 2.35 fold increased risk of
atrial fibrillation compared to carriers of the PAR1 variants
C3329A/A3329A. In carriers of the PAR1 variant C3329C, said variant
seems, in addition, to be protective with respect to the appearance
of acute coronary syndrome and unstable angina. Carriers of the
PAR1 variant C3329C have a 2.78 fold reduced risk of the appearance
of acute coronary syndrome and/or unstable angina compared to
carriers of the PAR1 variants A3329C/A3329A (FIG. 4).
[0141] It is therefore possible, by means of a method of the
invention and using an isolated PAR1 sequence of the particular SNP
type or a fragment thereof, to determine for human individuals
whether there is as assignment a risk group in accordance with the
results presented.
[0142] Preparation of Plasmid DNA
[0143] 1 ml of a bacterial overnight culture is transferred to an
Eppendorf tube and centrifuged (5 000 rpm for 5 min) in a Heraeus
Biofuge. The bacterial cell pellet is to be resuspended in 100
.mu.l of cooled solution I and then to be placed on ice for 5
min.
[0144] Solution I: 25 mM tris-HCl, pH 8.0, 50 mM glucose
(sterile-filtered) 10 mM EDTA 100 .mu.g/ml Rnase A.
[0145] After addition of 200 .mu.l of solution II, the entire
mixture is mixed well, resulting in alkaline denaturation of the
DNA.
[0146] Solution Il: 200 mM NaOH, 1% SDS.
[0147] After subsequent incubation for 5 min on ice, 150 p of
solution III are added to the mixture. This is followed by mixing
once more and incubating on ice for a further 15 min.
[0148] Solution III: 3 M sodium acetate (pH 4.8).
[0149] Centrifugation in the Heraeus Biofuge at 12 000 rpm for 15
minutes removes the cell debris, the genomic DNA and the denatured
proteins. The supernatant produced, which contains the plasmid DNA,
is decanted into a second Eppendorf tube and admixed with 1 ml of
96% strength EtOH (or 300 .mu.l of isopropanol). The precipitation
mixture is mixed thoroughly and again centrifuged (15 min at 12 000
rpm in Heraeus Biofuge). This results in precipitation of the
plasmid DNA. The plasmid DNA sediment is washed with ice-cold 70%
strength EtOH and then dried in air. Finally, the dry sediment is
taken up in 50 .mu.l of sterile distilled water.
[0150] Alcohol Precipitation of DNA
[0151] Precipitation mixture: DNA solution, {fraction (1/10)}
volume of 3 M sodium acetate (pH 5.4), 2 to 3 volumes of 96% EtOH
(1 volume of isopropanol).
[0152] The mixture is mixed well and can be stored at -20.degree.
C., although this does not increase the precipitation yield. The
plasmid DNA is sedimented by centrifugation at 12 000 rpm for 20
minutes.
[0153] In order to remove residues of the sodium acetate used, the
plasmid DNA must be washed once more with 1 ml of 70% strength EtOH
after precipitation.
[0154] Phenol Extraction of DNA
[0155] A DNA solution is admixed with the same volume of phenol
(Rotiphenol.RTM., equilibrated with TE buffer, pH 7.6, Roth,
Karlsruhe, Germany), shaken for 5 min and centrifuged at 5000 rpm.
Most of the now denatured proteins accumulate in the interface. The
upper, aqueous phase contains the DNA and is carefully removed by
suction, and then mixed with a chloroform/isoamyl alcohol mixture
(24:1) in order to remove phenol residues. This is followed by
another centrifugation, after which the aqueous supernatant is
removed and the DNA is isolated from the solution by alcohol
precipitation.
[0156] Purification of Amplified DNA Molecules
[0157] DNA amplicons are purified using a PCR purification kit
(Qiagen). This removes the starter molecules, nucleotides (dNTPs),
polymerases and salts. For this purpose, the PCR reaction mixture
is admixed with five times the volume of PB buffer, mixed well and
applied to the Qiaquick column. The amplified DNA is then
selectively bound to the column material, and the dNPTs are removed
by washing twice with 750 .mu.l of PE buffer. The amplified DNA is
then eluted with the desired volume of water, with the best volume
being the same as that of the PCR reaction mixture starting
material.
[0158] DNA Cleavage with Restriction Enzymes
[0159] Mix: 3 .mu.l of DNA, 2 .mu.l of 10.times.cleavage buffer,
2.5-5 U of restriction enzyme (e.g. EcORI, BamHI, SalI, XbaI, XhoI
etc.), add distilled water to a volume of 20 .mu.l.
[0160] Depending on the restriction enzyme, the cleavage reaction
runs at 25-55.degree. C. for 1-2 h. For analysis, the fragments are
electrophoretically fractionated in an agarose or polyacrylamide
gel in parallel with a length standard. If the reaction is a double
cleavage, then first one enzyme is added to the mixture. After 1
hour, an aliquot is applied to an appropriate gel, and, if the
cleavage has occurred, the second enzyme can be added. If the
second enzyme does not cleave in the same cleavage buffer, then an
alcohol precipitation is required first.
[0161] Agarose Gel Electrophoresis of DNA
[0162] The agarose (Roth) is dissolved in 1.times.agarose buffer at
the desired concentration and boiled in a microwave oven, until the
agarose has completely dissolved. The solution is then poured into
a sealed Plexiglass flat bed gel chamber.
[0163] The DNA samples are admixed with {fraction (1/10)} volume of
loading blue (50% v/v glycerol; 50 mM EDTA; 0.005% w/v BPB [Merck,
Darmstadt, Germany] and 0.005% xylene cyanol) and pipetted into the
gel pockets which are generated by means of a comb.
[0164] The electrophoresis is carried out horizontally in 1.times.
agarose buffer as running buffer at a constant voltage of 80-140 V,
depending on the size of the gel and the distance between the
electrodes.
[0165] 1.times.agarose buffer: 40 mM Tris-HCl (pH 7.8), 5 mM sodium
acetate, 1 mM EDTA.
[0166] Polyacrylamide Gel Electrophoresis of DNA
[0167] 7.5% polyacrylamide gel solution; 0.94 ml of 40% strength
acrylamide-bisacrylamide stock solution, 0.5 ml of 10.times.TBE
buffer (400 mM Tris-HCl, pH 8.3; 200 mM sodium acetate, 20 mM
EDTA), 0.25 ml of 1% AMPS, 10 .mu.l of TEMED, 3.33 ml of distilled
water.
[0168] This mixture is poured between well-cleaned, vertical glass
plates mounted in vertical apparatuses for polymerization (approx.
10-20 min). The gel is run in 1.times.TBE buffer at a constant
voltage of 104 V.
[0169] DNA Sequencing
[0170] 1-2 .mu.g of DNA are to be dissolved in 81 .mu.l of
distilled H.sub.2O and 9 .mu.l of NaOH (2 N) is to be added for
denaturation. After incubation at room temperature for 10 minutes,
the mixture is precipitated, with thorough washing of the resulting
DNA sediment with ice-cold 80% strength ethanol being important for
the subsequent sequencing reactions. 2 .mu.l of 5.times.Sequenase
buffer (200 mM Tris-Cl pH 7.5/100 mM MgCl.sub.2/250 mM NaCl), 1
.mu.l of oligonucleotide (1 .mu.M/.mu.l) and, finally, distilled
H.sub.2O are to be added to the sediment to a total volume of 10
.mu.l. During the subsequent incubation in a 37.degree. C. water
bath for 30 minutes, the starter oligonucleotide hybridizes to the
DNA.
[0171] Reagents added to the hybridization mixture for the
sequencing reaction: 1.0 .mu.l of DTT (0.1 M), 2.0 .mu.l of
labeling mixture (diluted 1:5), 0.5 .mu.l of
[.alpha.-.sup.35S]dATP, 2 .mu.l of Sequenase.TM. (13 U/.mu.l,
United States Biochemical), (diluted 1:8 with enzyme dilution
buffer).
[0172] During the subsequent incubation at room temperature for 5
minutes, the counter strand is synthesized, with the synthetic DNA
being labled by incorporation of the radiolabeled dATP. This is
followed by adding in each case 3.5 .mu.l of the labeling mixture
to 2.5 .mu.l of the four different termination mixtures. Another
incubation at 37.degree. C. for 5 minutes results in the randomly
distributed termination reactions of counter stand synthesis. The
reactions are stopped by adding 4 .mu.l of stop buffer, after which
the mixtures are denatured at 80-90.degree. C. and then applied to
a 6% strength denatured sequencing gel. After loading the samples,
the main run is carried out at 30-50 V and, respectively, 1300-1600
V for 2-5 h. The gel is then fixed in a 10% strength acetic acid
bath (15 min), freed of urea residues under running water and then
dried (for 45 min, using a heat gun, or for 2 h, in a 70.degree. C.
incubator). The subsequent autoradiography is carried out at
4.degree. C. for 16-24 h (Fuji Medical X-ray-Film RX, 30.times.40;
Kodak Scientific Imaging Film X-omat AR).
[0173] Labeling-mixture stock solution: in each case 7.5 .mu.M
dATP, dTTP, dGTP, dCTP
[0174] Termination mixtures: in each case 80 .mu.M dATP, dTTP,
dGTP, dCTP and in each case 8 .mu.M of the respective ddNTP
[0175] Sequenase dilution buffer: 10 mM Tris/HCl; pH 7.5, 5 mM DTT,
0.5 mg/ml BSA
[0176] Stop buffer: 95% formamide, 20 mM EDTA, 0.005% (w/v) xylene
cyanol FF
[0177] Automated DNA Sequencing
[0178] Mix: 1 .mu.g of plasmid DNA (in the case of PCR fragments,
for example, 100ng/500 nucleotides), 3-5 pmol of starter molecule
(PCR primer, Tm of 55.degree. C., if possible), 4 .mu.l of Dye
Terminator ready-mix (FddNTPs-Ampli-TaqFS mixture), add distilled
water to a volume of 20 .mu.l.
[0179] The PCR reaction [25.times.(15 sec at 94.degree. C., 15 sec.
at 50.degree. C., 4 min at 60.degree. C. is precipitated with
alcohol and taken up in 4 .mu.l of loading buffer. The samples are
then denatured at 95.degree. C. for 3 min, removed by
centrifugation and applied to a vertical polyacrylamide gel (34 cm
in length, provided with 24 parallel lanes).
[0180] After excitation by an argon laser beam at 488 nm, the dyes
emit light of different wavelengths of between 525 nm and 605 nm
which is separated into its spectral colors via a grating, a
"spectrograph". The spectral colors are subsequently detected
simultaneously with the aid of the high-resolution pixel field of a
CCD camera. The data are recorded with the aid of a computer
(Macintosh Quadra/650 Macllcx Apple Share) and the corresponding
data analysis software (PE Biosystems, Weiterstadt, Germany).
[0181] Sequencing gel: 30 g of urea (Sigma), 21.5 ml of distilled
H.sub.2O, 6 ml of 10.times.TBE
[0182] The mixture is dissolved in a wide-necked flask on a heating
block at 50.degree. C., with the following being added: 9 ml of 40%
bisacrylamide (filtered), 180 .mu.l 10 of 10% APS, 24 .mu.l of
Temed.
[0183] Polymerase Chain Reaction (PCR Reaction)
[0184] The following DNA polymerases may be used:
[0185] Taq (Thermus aquaticus) DNA polymerase (recombinant,
Gibco/BRL) and
[0186] 10.times.PCR buffer
[0187] [200 mM Tris/HCl (pH 8.4), 500 mM KCl]
[0188] Tfl (Thermus flavus) DNA polymerase (Master Amp.TM., Biozym,
Oldendorf, Germany) and 20.times.PCR
[0189] Buffer [20 mM (NH.sub.2)SO.sub.4, 1 M Tris/HCl (pH 9.0)
[0190] PCR Reaction Mixture:
4 PCR components Amount DNA template 10-100 ng Starter molecule 1
25 .mu.M Starter molecule 2 25 .mu.M Nucleotide mixture (dNTPs) 20
mM (from a mixture containing 10 mM of each dNTP) DNA polymerase
buffer 1.times.: 5.0 .mu.l in the case of Taq DNA polymerase buffer
2.5 .mu.l in the case of Tfl DNA polymerase buffer MgCl.sub.2 75 mM
DNA polymerase 2 U in the case of Taq DNA polymerase 1 U in the
case of Tfl DNA polymerase Distilled H.sub.2O to 50 .mu.l total
volume
[0191] The following applies here: 1 U catalyses the conversion of
10 nM deoxyribonucleoside triphosphates, at 74.degree. C. within 30
min, to an acid-insoluble DNA product. The PCR reaction usually
commences with the "hot start": the mixture is incubated first
without the polymerase at 94.degree. C. in order to enable the DNA
to be denatured for the first time. After the temperature has
reached 80.degree. C., the DNA polymerase is added to the mixture
in order to avoid nonspecific amplification at a still low
temperature. Thereafter, the actual PCR reaction is carried out
over 25-35 cycles.
[0192] For each cycle, the following reaction conditions apply:
5 Reaction Temperature Time Denaturation 94.degree. C. 30-60 sec
Hybridization T.sub.m-5.degree. C. 30-60 sec (annealing) Extension
72.degree. C. 1 min/1 kb
[0193] Finally and in addition, the chain extension is carried out
at 72.degree. C. for 10 min, finally followed by cooling.
[0194] Isolation of Total RNA
[0195] All centrifugation steps are carried out at 13 000 rpm and
16.degree. C. Cells are lysed with 600 .mu.l of lysis buffer (100
RLT buffer: 1 mercaptoethanol). The cell lysate is applied to a
QiaSchredder column and removed by centrifugation for 2 min.
[0196] The eluate is admixed with 600 .mu.l of 70% ethanol, mixed
well, and the DNA is applied to an RNAeasy mini spin column and
centrifuged for 15 s (binding of RNA to the silica matrix). The
column is washed three times (once with 700 ml of RW1 buffer and
twice with 500 .mu.l of RPE buffer). The column is then transferred
to an autoclaved 1.5 ml Eppendorf tube and the RNA is eluated with
15 .mu.l of distilled H.sub.2O. The average concentration of total
RNA obtained in this way is 1 .mu.g/.mu.l.
[0197] RNA Fractionation via Agarose Gel Electrophoresis
[0198] Denaturing aragrose gel:
[0199] 1 g of agraose, 37 ml of distilled water, 10 ml of
10.times.MOPS (0.2 mM
[0200] MOPS, 10 mM EDTA, 100 mM NaAc),
[0201] the mixture is boiled and cooled to 60.degree. C.
[0202] 16 ml of 37% strength formaldehyde are added.
[0203] After it has solidified, the gel is inserted with RNA gel
running buffer into the electrophoresis apparatus. The RNA is
applied together with a special sample buffer.
[0204] RNA gel running buffer: 40 ml of 10.times.MOPS, 65 ml of 37%
strength formaldehyde, 295 ml of distilled water
[0205] RNA sample buffer: 1-5 .mu.g of RNA, 5 .mu.l of RNA-NEW
buffer (7.5 .mu.l 37% strength formaldehyde, 4.5 .mu.l of
10.times.MOPS, 25.9 .mu.l of formamide, 7.5 .mu.l of distilled
water), 2 .mu.l of formamide dye marker [50% (v/v) glycerol, 1 mM
EDTA (pH 8.0), 0.25% (v/v) bromophenol blue, 0.25% (v/v) xylene
cyanol].
[0206] The gel runs at 80 V for approx. 3 h. Since this work uses
only eukaryotic RNA isolates, the dominant bands visible on the gel
should be those of 28S and 18S rRNA.
[0207] Reverse Transcriptase with MMLV-RT
[0208] (Moloney murine leukemia virus--Reverse Transcriptase)
[0209] Reverse transcriptase mixture: 5 .mu.g of RNA, 100 .mu.M of
starter molecule
[0210] The RNA preparation and the starter molecule are incubated
at 75.degree. C. for 10 min, in order to avoid possible formation
of secondary structures in the RNA template as factors interfering
with the transcriptase. However, even without this step, a
transcription reaction usually takes place.
[0211] Reverse transcriptase reaction mixture: 28 U of Rnasine
(Promega), 25 mM dNTPS, 5 .mu.l of 10.times. reverse transcriptase
buffer [10 m Tris/HCl (pH 8.3), 75 mM KCl, 3 mM MgCl.sub.2], 50 U
of reverse transcriptase (StrataScrip.TM., Stratagene), to 50 .mu.l
with distilled water.
[0212] The reverse transcription is carried out by incubating the
mixture at 42.degree. C. for 15 min and at 37.degree. C. for 45
min. A longer incubation of 2 h at 42.degree. C. with a 30-sec
interruption at 55.degree. C. is recommended for relatively long
RNA templates. Subsequent incubation of the mixture at 95.degree.
C. for 5 minutes results in inactivation of said reverse
transcriptase. Subsequently, 5-20 .mu.l of the reverse
transcription mixture are used for a PCR reaction.
[0213] Preparation of Genomic DNA from Tissue
[0214] 100 mg of tissue are crushed in liquid nitrogen to give a
powder. The tissue powder is introduced into a Falcon tube
containing 6 ml of reaction buffer (30 .mu.l of proteinase K [20
mg/ml] are added freshly to the buffer) and incubated with careful
shaking at 56.degree. C. overnight (12-18 hours). After incubation,
100 .mu.l of RNase A (10 .mu.g/.mu.l) are added and the mixture is
incubated with further shaking at 37.degree. C. for one hour.
[0215] This is followed by adding 4 ml of phenol and turning the
tube manually upside down and up again for approximately 5 min. 4
ml of Cl (chloroform/isoamyl alcohol) are added immediately and the
tube is turned upside down and up again for another 5 minutes and
then centrifuged for 15 min (3 000 rpm). The supernatant is
carefully removed and transferred to 10 ml Falcon tubes. If the
supernatant is still not clear, the phenol extraction must be
repeated, otherwise another 4 ml of Cl are added and the tube is
manually turned upside down and up for 5 min and then centrifuged
for 15 min (3 000 rpm). The supernatant is carefully removed and
the Cl extraction repeated. The final supernatant obtained is
admixed with {fraction (1/10)} volumes of sodium acetate solution
(3 M, pH 6) and 2.5 volumes of ethanol (99.8%). The tube is
carefully rotated, until the DNA precipitates as a tangle. This DNA
tangle is transferred to approximately 25 ml of ethanol (70%) with
the aid of a glass hook and left resting for 3 min. The washing was
repeated twice. The DNA was then dried in air and dissolved in 0.5
ml of double-distilled water at room temperature.
[0216] Southern Blot
[0217] DNA Fractionation via an Agarose Gel
[0218] Leave the gel on short-wave UV for approx. 5 min for strand
breaks to occur in the larger DNA molecules (>6 kBp).
[0219] Continuously tilt the gel in denaturating solution for 30
min for DNA denaturation.
[0220] Continuously tilt the gel in neutralizing solution for 30
min for neutralization.
[0221] Blot construction (from bottom to top): gel, nylon membrane,
dry filter paper, blotting paper, plate, weight (approx. 1 kg).
[0222] Blotting with 20.times.SCC overnight.
[0223] Wash membrane in 2.times.SSC for 10 min
[0224] Dry membrane on filter paper
[0225] Fixing of nucleic acid by baking at 80.degree. C. for 1 h or
UV crosslinking (e.g. in "Stratalinker", automatic position). The
membrane may then be stored until hybridization.
[0226] Prehybridization of membrane in hybridization solution for
approx. 1-2 h Covering of nonspecific binding sites on the
membrane.
[0227] Hybridization solution: 5.times.SSC, 5.times. Denhardt's
solution, 0.5% SDS, 100 .mu.g/ml herring sperm DNA
[0228] Denaturing solution: 0.5 M NaOH (20 g), 1 M NaCl
[0229] Neutralizing solution: 1.5 M NaCl/0.5 M Tris pH 7.4
[0230] 20.times.SSC is 3 M NaCl, 0.3 M Na-citrate: 175.3 g of NaCl,
88.2 g of sodium citrate.times.2H.sub.2O, to 1 1 with
double-distilled water, adjust pH to 7.0 with HCl.
[0231] 50.times. Denhard's solution: 5 g of Ficoll 400, 5 g of PVP
(polyvinyl pyrrolidone), 5 g of BSA, to 500 ml with
double-distilled water
[0232] Northern Blot
[0233] RNA Fractionation using a Formaldehyde Agarose Gel
[0234] Blot construction (from bottom to top): gel, nylon membrane,
dry filter paper, blotting paper, plate, weight (appox. 1 kg).
[0235] Blotting with 20.times.SSC overnight.
[0236] Fix RNA on filter by baking at 80.degree. C. (1 h)
[0237] Introduce filter into boiling 20 mM Tris pH 8 for RNA
deglyoxylation and let cool to RT.
Sequence CWU 1
1
13 1 3592 DNA Homo sapiens 1 ggcggggggc gcacagagcc agaggggctt
gcgagcggcg gctgagggac cgcggggagg 60 gggcgccgag cggctccagc
gcagagactc tcactgcacg ccggaggccc cttcctcgct 120 ccgcccgcgc
gaccgcgcgc cccagtcccg ccccgccccg ctaaccgccc cagacacagc 180
gctcgccgag ggtcgcttgg accctgatct tacccgtggg caccctgcgc tctgcctgcc
240 gcgaagaccg gctccccgac ccgcagaagt caggagagag ggtgaagcgg
agcagcccga 300 ggcggggcag cctcccggag cagcgccgcg cagagcccgg
gacaatgggg ccgcggcggc 360 tgctgctggt ggccgcctgc ttcagtctgt
gcggcccgct gttgtctgcc cgcacccggg 420 cccgcaggcc agaatcaaaa
gcaacaaatg ccaccttaga tccccggtca tttcttctca 480 ggaaccccaa
tgataaatat gaaccatttt gggaggatga ggagaaaaat gaaagtgggt 540
taactgaata cagattagtc tccatcaata aaagcagtcc tcttcaaaaa caacttcctg
600 cattcatctc agaagatgcc tccggatatt tgaccagctc ctggctgaca
ctctttgtcc 660 catctgtgta caccggagtg tttgtagtca gcctcccact
aaacatcatg gccatcgttg 720 tgttcatcct gaaaatgaag gtcaagaagc
cggcggtggt gtacatgctg cacctggcca 780 cggcagatgt gctgtttgtg
tctgtgctcc cctttaagat cagctattac ttttccggca 840 gtgattggca
gtttgggtct gaattgtgtc gcttcgtcac tgcagcattt tactgtaaca 900
tgtacgcctc tatcttgctc atgacagtca taagcattga ccggtttctg gctgtggtgt
960 atcccatgca gtccctctcc tggcgtactc tgggaagggc ttccttcact
tgtctggcca 1020 tctgggcttt ggccatcgca ggggtagtgc ctctcgtcct
caaggagcaa accatccagg 1080 tgcccgggct caacatcact acctgtcatg
atgtgctcaa tgaaaccctg ctcgaaggct 1140 actatgccta ctacttctca
gccttctctg ctgtcttctt ttttgtgccg ctgatcattt 1200 ccacggtctg
ttatgtgtct atcattcgat gtcttagctc ttccgcagtt gccaaccgca 1260
gcaagaagtc ccgggctttg ttcctgtcag ctgctgtttt ctgcatcttc atcatttgct
1320 tcggacccac aaacgtcctc ctgattgcgc attactcatt cctttctcac
acttccacca 1380 cagaggctgc ctactttgcc tacctcctct gtgtctgtgt
cagcagcata agctcgtgca 1440 tcgaccccct aatttactat tacgcttcct
ctgagtgcca gaggtacgtc tacagtatct 1500 tatgctgcaa agaaagttcc
gatcccagca gttataacag cagtgggcag ttgatggcaa 1560 gtaaaatgga
tacctgctct agtaacctga ataacagcat atacaaaaag ctgttaactt 1620
aggaaaaggg actgctggga ggttaaaaag aaaagtttat aaaagtgaat aacctgagga
1680 ttctattagt ccccacccaa actttattga ttcacctcct aaaacaacag
atgtacgact 1740 tgcatacctg ctttttatgg gagctgtcaa gcatgtattt
ttgtcaatta ccagaaagat 1800 aacaggacga gatgacggtg ttattccaag
ggaatattgc caatgctaca gtaataaatg 1860 aatgtcactt ctggatatag
ctaggtgaca tatacatact tacatgtgtg tatatgtaga 1920 tgtatgcaca
cacatatatt atttgcagtg cagtatagaa taggcacttt aaaacactct 1980
ttccccgcac cccagcaatt atgaaaataa tctctgattc cctgatttaa tatgcaaagt
2040 ctaggttggt agagtttagc cctgaacatt tcatggtgtt catcaacagt
gagagactcc 2100 atagtttggg cttgtaccac ttttgcaaat aagtgtattt
tgaaattgtt tgacggcaag 2160 gtttaagtta ttaagaggta agacttagta
ctatctgtgc gtagaagttc tagtgttttc 2220 aattttaaac atatccaagt
ttgaattcct aaaattatgg aaacagatga aaagcctctg 2280 ttttgatatg
ggtagtattt tttacatttt acacactgta cacataagcc aaaactgagc 2340
ataagtcctc tagtgaatgt aggctggctt tcagagtagg ctattcctga gagctgcatg
2400 tgtccgcccc cgatggagga ctccaggcag cagacacatg ccagggccat
gtcagacaca 2460 gattggccag aaaccttcct gctgagcctc acagcagtga
gactggggcc actacatttg 2520 ctccatcctc ctgggattgg ctgtgaactg
atcatgttta tgagaaactg gcaaagcaga 2580 atgtgatatc ctaggaggta
atgaccatga aagacttctc tacccatctt aaaaacaacg 2640 aaagaaggca
tggacttctg gatgcccatc cactgggtgt aaacacatct agtagttgtt 2700
ctgaaatgtc agttctgata tggaagcacc cattatgcgc tgtggccact ccaataggtg
2760 ctgagtgtac agagtggaat aagacagaga cctgccctca agagcaaagt
agatcatgca 2820 tagagtgtga tgtatgtgta ataaatatgt ttcacacaaa
caaggcctgt cagctaaaga 2880 agtttgaaca tttgggttac tatttcttgt
ggttataact taatgaaaac aatgcagtac 2940 aggacatata ttttttaaaa
taagtctgat ttaattgggc actatttatt tacaaatgtt 3000 ttgctcaata
gattgctcaa atcaggtttt cttttaagaa tcaatcatgt cagtctgctt 3060
agaaataaca gaagaaaata gaattgacat tgaaatctag gaaaattatt ctataatttc
3120 catttactta agacttaatg agactttaaa agcatttttt aacctcctaa
gtatcaagta 3180 tagaaaatct tcatggaatt cacaaagtaa tttggaaatt
aggttgaaac atatctctta 3240 tcttacgaaa aaatggtagc attttaaaca
aaatagaaag ttgcaaggca aatgtttatt 3300 taaaagagca ggccaggcgc
ggtggctcac gcctgtaatc ccagcacttt gggaggctga 3360 ggcgggtgga
tcacgaggtc aggagatcga gaccatcctg gctaacacgg tgaaacccgt 3420
ctctactaaa aatgcaaaaa aaattagccg ggcgtggtgg caggcacctg tagtcccagc
3480 tactcgggag gctgaggcag gagactggcg tgaacccagg aggcggacct
tgtagtgagc 3540 cgagatcgcg ccactgtgct ccagcctggg caacagagca
agactccatc tc 3592 2 3592 DNA Homo sapiens 2 ggcggggggc gcacagagcc
agaggggctt gcgagcggcg gctgagggac cgcggggagg 60 gggcgccgag
cggctccagc gcagagactc tcactgcacg ccggaggccc cttcctcgct 120
ccgcccgcgc gaccgcgcgc cccagtcccg ccccgccccg ctaaccgccc cagacacagc
180 gctcgccgag ggtcgcttgg accctgatct tacccgtggg caccctgcgc
tctgcctgcc 240 gcgaagaccg gctccccgac ccgcagaagt caggagagag
ggtgaagcgg agcagcccga 300 ggcggggcag cctcccggag cagcgccgcg
cagagcccgg gacaatgggg ccgcggcggc 360 tgctgctggt ggccgcctgc
ttcagtctgt gcggcccgct gttgtctgcc cgcacccggg 420 cccgcaggcc
agaatcaaaa gcaacaaatg ccaccttaga tccccggtca tttcttctca 480
ggaaccccaa tgataaatat gaaccatttt gggaggatga ggagaaaaat gaaagtgggt
540 taactgaata cagattagtc tccatcaata aaagcagtcc tcttcaaaaa
caacttcctg 600 cattcatctc agaagatgcc tccggatatt tgaccagctc
ctggctgaca ctctttgtcc 660 catctgtgta caccggagtg tttgtagtca
gcctcccact aaacatcatg gccatcgttg 720 tgttcatcct gaaaatgaag
gtcaagaagc cggcggtggt gtacatgctg cacctggcca 780 cggcagatgt
gctgtttgtg tctgtgctcc cctttaagat cagctattac ttttccggca 840
gtgattggca gtttgggtct gaattgtgtc gcttcgtcac tgcagcattt tactgtaaca
900 tgtacgcctc tatcttgctc atgacagtca taagcattga ccggtttctg
gctgtggtgt 960 atcccatgca gtccctctcc tggcgtactc tgggaagggc
ttccttcact tgtctggcca 1020 tctgggcttt ggccatcgca ggggtagtgc
ctctcgtcct caaggagcaa accatccagg 1080 tgcccgggct caacatcact
acctgtcatg atgtgctcaa tgaaaccctg ctcgaaggct 1140 actatgccta
ctacttctca gccttctctg ctgtcttctt ttttgtgccg ctgatcattt 1200
ccacggtctg ttatgtgtct atcattcgat gtcttagctc ttccgcagtt gccaaccgca
1260 gcaagaagtc ccgggctttg ttcctgtcag ctgctgtttt ctgcatcttc
atcatttgct 1320 tcggacccac aaacgtcctc ctgattgcgc attactcatt
cctttctcac acttccacca 1380 cagaggctgc ctactttgcc tacctcctct
gtgtctgtgt cagcagcata agctcgtgca 1440 tcgaccccct aatttactat
tacgcttcct ctgagtgcca gaggtacgtc tacagtatct 1500 tatgctgcaa
agaaagttcc gatcccagca gttataacag cagtgggcag ttgatggcaa 1560
gtaaaatgga tacctgctct agtaacctga ataacagcat atacaaaaag ctgttaactt
1620 aggaaaaggg actgctggga ggttaaaaag aaaagtttat aaaagtgaat
aacctgagga 1680 ttctattagt ccccacccaa actttattga ttcacctcct
aaaacaacag atgtacgact 1740 tgcatacctg ctttttatgg gagctgtcaa
gcatgtattt ttgtcaatta ccagaaagat 1800 aacaggacga gatgacggtg
ttattccaag ggaatattgc caatgctaca gtaataaatg 1860 aatgtcactt
ctggatatag ctaggtgaca tatacatact tacatgtgtg tatatgtaga 1920
tgtatgcaca cacatatatt atttgcagtg cagtatagaa taggcacttt aaaacactct
1980 ttccccgcac cccagcaatt atgaaaataa tctctgattc cctgatttaa
tatgcaaagt 2040 ctaggttggt agagtttagc cctgaacatt tcatggtgtt
catcaacagt gagagactcc 2100 atagtttggg cttgtaccac ttttgcaaat
aagtgtattt tgaaattgtt tgacggcaag 2160 gtttaagtta ttaagaggta
agacttagta ctatctgtgc gtagaagttc tagtgttttc 2220 aattttaaac
atatccaagt ttgaattcct aaaattatgg aaacagatga aaagcctctg 2280
ttttgatatg ggtagtattt tttacatttt acacactgta cacataagcc aaaactgagc
2340 ataagtcctc tagtgaatgt aggctggctt tcagagtagg ctattcctga
gagctgcatg 2400 tgtccgcccc cgatggagga ctccaggcag cagacacatg
ccagggccat gtcagacaca 2460 gattggccag aaaccttcct gctgagcctc
acagcagtga gactggggcc actacatttg 2520 ctccatcctc ctgggattgg
ctgtgaactg atcatgttta tgagaaactg gcaaagcaga 2580 atgtgatatc
ctaggaggta atgaccatga aagacttctc tacccatctt aaaaacaacg 2640
aaagaaggca tggacttctg gatgcccatc cactgggtgt aaacacatct agtagttgtt
2700 ctgaaatgtc agttctgata tggaagcacc cattatgcgc tgtggccact
ccaataggtg 2760 ctgagtgtac agagtggaat aagacagaga cctgccctca
agagcaaagt agatcatgca 2820 tagagtgtga tgtatgtgta ataaatatgt
ttcacacaaa caaggcctgt cagctaaaga 2880 agtttgaaca tttgggttac
tatttcttgt ggttataact taatgaaaac aatgcagtac 2940 aggacatata
ttttttaaaa taagtctgat ttaattgggc actatttatt tacaaatgtt 3000
ttgctcaata gattgctcaa atcaggtttt cttttaagaa tcaatcatgt cagtctgctt
3060 agaaataaca gaagaaaata gaattgacac tgaaatctag gaaaattatt
ctataatttc 3120 catttactta agacttaatg agactttaaa agcatttttt
aacctcctaa gtatcaagta 3180 tagaaaatct tcatggaatt cacaaagtaa
tttggaaatt aggttgaaac atatctctta 3240 tcttacgaaa aaatggtagc
attttaaaca aaatagaaag ttgcaaggca aatgtttatt 3300 taaaagagca
ggccaggcgc ggtggctcac gcctgtaatc ccagcacttt gggaggctga 3360
ggcgggtgga tcacgaggtc aggagatcga gaccatcctg gctaacacgg tgaaacccgt
3420 ctctactaaa aatgcaaaaa aaattagccg ggcgtggtgg caggcacctg
tagtcccagc 3480 tactcgggag gctgaggcag gagactggcg tgaacccagg
aggcggacct tgtagtgagc 3540 cgagatcgcg ccactgtgct ccagcctggg
caacagagca agactccatc tc 3592 3 3592 DNA Homo sapiens 3 ggcggggggc
gcacagagcc agaggggctt gcgagcggcg gctgagggac cgcggggagg 60
gggcgccgag cggctccagc gcagagactc tcactgcacg ccggaggccc cttcctcgct
120 ccgcccgcgc gaccgcgcgc cccagtcccg ccccgccccg ctaaccgccc
cagacacagc 180 gctcgccgag ggtcgcttgg accctgatct tacccgtggg
caccctgcgc tctgcctgcc 240 gcgaagaccg gctccccgac ccgcagaagt
caggagagag ggtgaagcgg agcagcccga 300 ggcggggcag cctcccggag
cagcgccgcg cagagcccgg gacaatgggg ccgcggcggc 360 tgctgctggt
ggccgcctgc ttcagtctgt gcggcccgct gttgtctgcc cgcacccggg 420
cccgcaggcc agaatcaaaa gcaacaaatg ccaccttaga tccccggtca tttcttctca
480 ggaaccccaa tgataaatat gaaccatttt gggaggatga ggagaaaaat
gaaagtgggt 540 taactgaata cagattagtc tccatcaata aaagcagtcc
tcttcaaaaa caacttcctg 600 cattcatctc agaagatgcc tccggatatt
tgaccagctc ctggctgaca ctctttgtcc 660 catctgtgta caccggagtg
tttgtagtca gcctcccact aaacatcatg gccatcgttg 720 tgttcatcct
gaaaatgaag gtcaagaagc cggcggtggt gtacatgctg cacctggcca 780
cggcagatgt gctgtttgtg tctgtgctcc cctttaagat cagctattac ttttccggca
840 gtgattggca gtttgggtct gaattgtgtc gcttcgtcac tgcagcattt
tactgtaaca 900 tgtacgcctc tatcttgctc atgacagtca taagcattga
ccggtttctg gctgtggtgt 960 atcccatgca gtccctctcc tggcgtactc
tgggaagggc ttccttcact tgtctggcca 1020 tctgggcttt ggccatcgca
ggggtagtgc ctctcgtcct caaggagcaa accatccagg 1080 tgcccgggct
caacatcact acctgtcatg atgtgctcaa tgaaaccctg ctcgaaggct 1140
actatgccta ctacttctca gccttctctg ctgtcttctt ttttgtgccg ctgatcattt
1200 ccacggtctg ttatgtgtct atcattcgat gtcttagctc ttccgcagtt
gccaaccgca 1260 gcaagaagtc ccgggctttg ttcctgtcag ctgctgtttt
ctgcatcttc atcatttgct 1320 tcggacccac aaacgtcctc ctgattgcgc
attactcatt cctttctcac acttccacca 1380 cagaggctgc ctactttgcc
tacctcctct gtgtctgtgt cagcagcata agctcgtgca 1440 tcgaccccct
aatttactat tacgcttcct ctgagtgcca gaggtacgtc tacagtatct 1500
tatgctgcaa agaaagttcc gatcccagca gttataacag cagtgggcag ttgatggcaa
1560 gtaaaatgga tacctgctct agtaacctga ataacagcat atacaaaaag
ctgttaactt 1620 aggaaaaggg actgctggga ggttaaaaag aaaagtttat
aaaagtgaat aacctgagga 1680 ttctattagt ccccacccaa actttattga
ttcacctcct aaaacaacag atgtacgact 1740 tgcatacctg ctttttatgg
gagctgtcaa gcatgtattt ttgtcaatta ccagaaagat 1800 aacaggacga
gatgacggtg ttattccaag ggaatattgc caatgctaca gtaataaatg 1860
aatgtcactt ctggatatag ctaggtgaca tatacatact tacatgtgtg tatatgtaga
1920 tgtatgcaca cacatatatt atttgcagtg cagtatagaa taggcacttt
aaaacactct 1980 ttccccgcac cccagcaatt atgaaaataa tctctgattc
cctgatttaa tatgcaaagt 2040 ctaggttggt agagtttagc cctgaacatt
tcatggtgtt catcaacagt gagagactcc 2100 atagtttggg cttgtaccac
ttttgcaaat aagtgtattt tgaaattgtt tgacggcaag 2160 gtttaagtta
ttaagaggta agacttagta ctatctgtgc gtagaagttc tagtgttttc 2220
aattttaaac atatccaagt ttgaattcct aaaattatgg aaacagatga aaagcctctg
2280 ttttgatatg ggtagtattt tttacatttt acacactgta cacataagcc
aaaactgagc 2340 ataagtcctc tagtgaatgt aggctggctt tcagagtagg
ctattcctga gagctgcatg 2400 tgtccgcccc cgatggagga ctccaggcag
cagacacatg ccagggccat gtcagacaca 2460 gattggccag aaaccttcct
gctgagcctc acagcagtga gactggggcc actacatttg 2520 ctccatcctc
ctgggattgg ctgtgaactg atcatgttta tgagaaactg gcaaagcaga 2580
atgtgatatc ctaggaggta atgaccatga aagacttctc tacccatctt aaaaacaacg
2640 aaagaaggca tggacttctg gatgcccatc cactgggtgt aaacacatct
agtagttgtt 2700 ctgaaatgtc agttctgata tggaagcacc cattatgcgc
tgtggccact ccaataggtg 2760 ctgagtgtac agagtggaat aagacagaga
cctgccctca agagcaaagt agatcatgca 2820 tagagtgtga tgtatgtgta
ataaatatgt ttcacacaaa caaggcctgt cagctaaaga 2880 agtttgaaca
tttgggttac tatttcttgt ggttataact taatgaaaac aatgcagtac 2940
aggacatata ttttttaaaa taagtctgat ttaattgggc actatttatt tacaaatgtt
3000 ttgctcaata gattgctcaa atcaggtttt cttttaagaa tcaatcatgt
cagtctgctt 3060 agaaataaca gaagaaaata gaattgacat tgaaatctag
gaaaattatt ctataatttc 3120 catttactta agacttaatg agactttaaa
agcatttttt aacctcctaa gtatcaagta 3180 tagaaaatct tcatggaatt
cacaaagtaa tttggaaatt aggttgaaac atatctctta 3240 tcttacgaaa
aaatggtagc attttaaaca aaatagaaag ttgcaaggca aatgtttatt 3300
taaaagagca ggccaggcgc ggtggctccc gcctgtaatc ccagcacttt gggaggctga
3360 ggcgggtgga tcacgaggtc aggagatcga gaccatcctg gctaacacgg
tgaaacccgt 3420 ctctactaaa aatgcaaaaa aaattagccg ggcgtggtgg
caggcacctg tagtcccagc 3480 tactcgggag gctgaggcag gagactggcg
tgaacccagg aggcggacct tgtagtgagc 3540 cgagatcgcg ccactgtgct
ccagcctggg caacagagca agactccatc tc 3592 4 3592 DNA Homo sapiens 4
ggcggggggc gcacagagcc agaggggctt gcgagcggcg gctgagggac cgcggggagg
60 gggcgccgag cggctccagc gcagagactc tcactgcacg ccggaggccc
cttcctcgct 120 ccgcccgcgc gaccgcgcgc cccagtcccg ccccgccccg
ctaaccgccc cagacacagc 180 gctcgccgag ggtcgcttgg accctgatct
tacccgtggg caccctgcgc tctgcctgcc 240 gcgaagaccg gctccccgac
ccgcagaagt caggagagag ggtgaagcgg agcagcccga 300 ggcggggcag
cctcccggag cagcgccgcg cagagcccgg gacaatgggg ccgcggcggc 360
tgctgctggt ggccgcctgc ttcagtctgt gcggcccgct gttgtctgcc cgcacccggg
420 cccgcaggcc agaatcaaaa gcaacaaatg ccaccttaga tccccggtca
tttcttctca 480 ggaaccccaa tgataaatat gaaccatttt gggaggatga
ggagaaaaat gaaagtgggt 540 taactgaata cagattagtc tccatcaata
aaagcagtcc tcttcaaaaa caacttcctg 600 cattcatctc agaagatgcc
tccggatatt tgaccagctc ctggctgaca ctctttgtcc 660 catctgtgta
caccggagtg tttgtagtca gcctcccact aaacatcatg gccatcgttg 720
tgttcatcct gaaaatgaag gtcaagaagc cggcggtggt gtacatgctg cacctggcca
780 cggcagatgt gctgtttgtg tctgtgctcc cctttaagat cagctattac
ttttccggca 840 gtgattggca gtttgggtct gaattgtgtc gcttcgtcac
tgcagcattt tactgtaaca 900 tgtacgcctc tatcttgctc atgacagtca
taagcattga ccggtttctg gctgtggtgt 960 atcccatgca gtccctctcc
tggcgtactc tgggaagggc ttccttcact tgtctggcca 1020 tctgggcttt
ggccatcgca ggggtagtgc ctctcgtcct caaggagcaa accatccagg 1080
tgcccgggct caacatcact acctgtcatg atgtgctcaa tgaaaccctg ctcgaaggct
1140 actatgccta ctacttctca gccttctctg ctgtcttctt ttttgtgccg
ctgatcattt 1200 ccacggtctg ttatgtgtct atcattcgat gtcttagctc
ttccgcagtt gccaaccgca 1260 gcaagaagtc ccgggctttg ttcctgtcag
ctgctgtttt ctgcatcttc atcatttgct 1320 tcggacccac aaacgtcctc
ctgattgcgc attactcatt cctttctcac acttccacca 1380 cagaggctgc
ctactttgcc tacctcctct gtgtctgtgt cagcagcata agctcgtgca 1440
tcgaccccct aatttactat tacgcttcct ctgagtgcca gaggtacgtc tacagtatct
1500 tatgctgcaa agaaagttcc gatcccagca gttataacag cagtgggcag
ttgatggcaa 1560 gtaaaatgga tacctgctct agtaacctga ataacagcat
atacaaaaag ctgttaactt 1620 aggaaaaggg actgctggga ggttaaaaag
aaaagtttat aaaagtgaat aacctgagga 1680 ttctattagt ccccacccaa
actttattga ttcacctcct aaaacaacag atgtacgact 1740 tgcatacctg
ctttttatgg gagctgtcaa gcatgtattt ttgtcaatta ccagaaagat 1800
aacaggacga gatgacggtg ttattccaag ggaatattgc caatgctaca gtaataaatg
1860 aatgtcactt ctggatatag ctaggtgaca tatacatact tacatgtgtg
tatatgtaga 1920 tgtatgcaca cacatatatt atttgcagtg cagtatagaa
taggcacttt aaaacactct 1980 ttccccgcac cccagcaatt atgaaaataa
tctctgattc cctgatttaa tatgcaaagt 2040 ctaggttggt agagtttagc
cctgaacatt tcatggtgtt catcaacagt gagagactcc 2100 atagtttggg
cttgtaccac ttttgcaaat aagtgtattt tgaaattgtt tgacggcaag 2160
gtttaagtta ttaagaggta agacttagta ctatctgtgc gtagaagttc tagtgttttc
2220 aattttaaac atatccaagt ttgaattcct aaaattatgg aaacagatga
aaagcctctg 2280 ttttgatatg ggtagtattt tttacatttt acacactgta
cacataagcc aaaactgagc 2340 ataagtcctc tagtgaatgt aggctggctt
tcagagtagg ctattcctga gagctgcatg 2400 tgtccgcccc cgatggagga
ctccaggcag cagacacatg ccagggccat gtcagacaca 2460 gattggccag
aaaccttcct gctgagcctc acagcagtga gactggggcc actacatttg 2520
ctccatcctc ctgggattgg ctgtgaactg atcatgttta tgagaaactg gcaaagcaga
2580 atgtgatatc ctaggaggta atgaccatga aagacttctc tacccatctt
aaaaacaacg 2640 aaagaaggca tggacttctg gatgcccatc cactgggtgt
aaacacatct agtagttgtt 2700 ctgaaatgtc agttctgata tggaagcacc
cattatgcgc tgtggccact ccaataggtg 2760 ctgagtgtac agagtggaat
aagacagaga cctgccctca agagcaaagt agatcatgca 2820 tagagtgtga
tgtatgtgta ataaatatgt ttcacacaaa caaggcctgt cagctaaaga 2880
agtttgaaca tttgggttac tatttcttgt ggttataact taatgaaaac aatgcagtac
2940 aggacatata ttttttaaaa taagtctgat ttaattgggc actatttatt
tacaaatgtt 3000 ttgctcaata gattgctcaa atcaggtttt cttttaagaa
tcaatcatgt cagtctgctt 3060 agaaataaca gaagaaaata gaattgacac
tgaaatctag gaaaattatt ctataatttc 3120 catttactta agacttaatg
agactttaaa agcatttttt aacctcctaa gtatcaagta 3180 tagaaaatct
tcatggaatt cacaaagtaa tttggaaatt aggttgaaac atatctctta 3240
tcttacgaaa aaatggtagc attttaaaca aaatagaaag ttgcaaggca aatgtttatt
3300 taaaagagca ggccaggcgc ggtggctccc gcctgtaatc ccagcacttt
gggaggctga 3360 ggcgggtgga tcacgaggtc aggagatcga gaccatcctg
gctaacacgg tgaaacccgt 3420 ctctactaaa aatgcaaaaa aaattagccg
ggcgtggtgg caggcacctg tagtcccagc 3480 tactcgggag gctgaggcag
gagactggcg tgaacccagg aggcggacct tgtagtgagc 3540 cgagatcgcg
ccactgtgct ccagcctggg caacagagca agactccatc tc 3592 5 939 DNA Homo
sapiens 5 acagagtgga ataagacaga gacctgccct caagagcaaa gtagatcatg
catagagtgt 60 gatgtatgtg taataaatat gtttcacaca aacaaggcct
gtcagctaaa gaagtttgaa 120 catttgggtt actatttctt gtggttataa
cttaatgaaa acaatgcagt acaggacata 180 tattttttaa aataagtctg
atttaattgg gcactattta tttacaaatg ttttgctcaa 240 tagattgctc
aaatcaggtt ttcttttaag aatcaatcat gtcagtctgc ttagaaataa 300
cagaagaaaa tagaattgac attgaaatct aggaaaatta ttctataatt tccatttact
360 taagacttaa tgagacttta aaagcatttt ttaacctcct aagtatcaag
tatagaaaat 420 cttcatggaa ttcacaaagt aatttggaaa ttaggttgaa
acatatctct tatcttacga 480 aaaaatggta gcattttaaa caaaatagaa
agttgcaagg caaatgttta tttaaaagag
540 caggccaggc gcggtggctc acgcctgtaa tcccagcact ttgggaggct
gaggcgggtg 600 gatcacgagg tcaggagatc gagaccatcc tggctaacac
ggtgaaaccc gtctctacta 660 aaaatgcaaa aaaaattagc cgggcgtggt
ggcaggcacc tgtagtccca gctactcggg 720 aggctgaggc aggagactgg
cgtgaaccca ggaggcggac cttgtagtga gccgagatcg 780 cgccactgtg
ctccagcctg ggcaacagag caagactcca tctcaaaaaa taaaaataaa 840
taaaaaataa aaaaataaaa gagcaaacta tttccaaata ccatagaata acttacataa
900 aagtaatata actgtattgt aagtagaagc tagcactgg 939 6 939 DNA Homo
sapiens 6 acagagtgga ataagacaga gacctgccct caagagcaaa gtagatcatg
catagagtgt 60 gatgtatgtg taataaatat gtttcacaca aacaaggcct
gtcagctaaa gaagtttgaa 120 catttgggtt actatttctt gtggttataa
cttaatgaaa acaatgcagt acaggacata 180 tattttttaa aataagtctg
atttaattgg gcactattta tttacaaatg ttttgctcaa 240 tagattgctc
aaatcaggtt ttcttttaag aatcaatcat gtcagtctgc ttagaaataa 300
cagaagaaaa tagaattgac actgaaatct aggaaaatta ttctataatt tccatttact
360 taagacttaa tgagacttta aaagcatttt ttaacctcct aagtatcaag
tatagaaaat 420 cttcatggaa ttcacaaagt aatttggaaa ttaggttgaa
acatatctct tatcttacga 480 aaaaatggta gcattttaaa caaaatagaa
agttgcaagg caaatgttta tttaaaagag 540 caggccaggc gcggtggctc
acgcctgtaa tcccagcact ttgggaggct gaggcgggtg 600 gatcacgagg
tcaggagatc gagaccatcc tggctaacac ggtgaaaccc gtctctacta 660
aaaatgcaaa aaaaattagc cgggcgtggt ggcaggcacc tgtagtccca gctactcggg
720 aggctgaggc aggagactgg cgtgaaccca ggaggcggac cttgtagtga
gccgagatcg 780 cgccactgtg ctccagcctg ggcaacagag caagactcca
tctcaaaaaa taaaaataaa 840 taaaaaataa aaaaataaaa gagcaaacta
tttccaaata ccatagaata acttacataa 900 aagtaatata actgtattgt
aagtagaagc tagcactgg 939 7 939 DNA Homo sapiens 7 acagagtgga
ataagacaga gacctgccct caagagcaaa gtagatcatg catagagtgt 60
gatgtatgtg taataaatat gtttcacaca aacaaggcct gtcagctaaa gaagtttgaa
120 catttgggtt actatttctt gtggttataa cttaatgaaa acaatgcagt
acaggacata 180 tattttttaa aataagtctg atttaattgg gcactattta
tttacaaatg ttttgctcaa 240 tagattgctc aaatcaggtt ttcttttaag
aatcaatcat gtcagtctgc ttagaaataa 300 cagaagaaaa tagaattgac
attgaaatct aggaaaatta ttctataatt tccatttact 360 taagacttaa
tgagacttta aaagcatttt ttaacctcct aagtatcaag tatagaaaat 420
cttcatggaa ttcacaaagt aatttggaaa ttaggttgaa acatatctct tatcttacga
480 aaaaatggta gcattttaaa caaaatagaa agttgcaagg caaatgttta
tttaaaagag 540 caggccaggc gcggtggctc ccgcctgtaa tcccagcact
ttgggaggct gaggcgggtg 600 gatcacgagg tcaggagatc gagaccatcc
tggctaacac ggtgaaaccc gtctctacta 660 aaaatgcaaa aaaaattagc
cgggcgtggt ggcaggcacc tgtagtccca gctactcggg 720 aggctgaggc
aggagactgg cgtgaaccca ggaggcggac cttgtagtga gccgagatcg 780
cgccactgtg ctccagcctg ggcaacagag caagactcca tctcaaaaaa taaaaataaa
840 taaaaaataa aaaaataaaa gagcaaacta tttccaaata ccatagaata
acttacataa 900 aagtaatata actgtattgt aagtagaagc tagcactgg 939 8 939
DNA Homo sapiens 8 acagagtgga ataagacaga gacctgccct caagagcaaa
gtagatcatg catagagtgt 60 gatgtatgtg taataaatat gtttcacaca
aacaaggcct gtcagctaaa gaagtttgaa 120 catttgggtt actatttctt
gtggttataa cttaatgaaa acaatgcagt acaggacata 180 tattttttaa
aataagtctg atttaattgg gcactattta tttacaaatg ttttgctcaa 240
tagattgctc aaatcaggtt ttcttttaag aatcaatcat gtcagtctgc ttagaaataa
300 cagaagaaaa tagaattgac actgaaatct aggaaaatta ttctataatt
tccatttact 360 taagacttaa tgagacttta aaagcatttt ttaacctcct
aagtatcaag tatagaaaat 420 cttcatggaa ttcacaaagt aatttggaaa
ttaggttgaa acatatctct tatcttacga 480 aaaaatggta gcattttaaa
caaaatagaa agttgcaagg caaatgttta tttaaaagag 540 caggccaggc
gcggtggctc ccgcctgtaa tcccagcact ttgggaggct gaggcgggtg 600
gatcacgagg tcaggagatc gagaccatcc tggctaacac ggtgaaaccc gtctctacta
660 aaaatgcaaa aaaaattagc cgggcgtggt ggcaggcacc tgtagtccca
gctactcggg 720 aggctgaggc aggagactgg cgtgaaccca ggaggcggac
cttgtagtga gccgagatcg 780 cgccactgtg ctccagcctg ggcaacagag
caagactcca tctcaaaaaa taaaaataaa 840 taaaaaataa aaaaataaaa
gagcaaacta tttccaaata ccatagaata acttacataa 900 aagtaatata
actgtattgt aagtagaagc tagcactgg 939 9 20 DNA Homo sapiens 9
ggcggggggc gcacagagcc 20 10 22 DNA Homo sapiens 10 gagatggagt
cttgctctgt tg 22 11 21 DNA Homo sapiens 11 acagagtgga ataagacaga g
21 12 21 DNA Homo sapiens 12 ccagtgctag cttctactta c 21 13 425 PRT
Homo sapiens 13 Met Gly Pro Arg Arg Leu Leu Leu Val Ala Ala Cys Phe
Ser Leu Cys 1 5 10 15 Gly Pro Leu Leu Ser Ala Arg Thr Arg Ala Arg
Arg Pro Glu Ser Lys 20 25 30 Ala Thr Asn Ala Thr Leu Asp Pro Arg
Ser Phe Leu Leu Arg Asn Pro 35 40 45 Asn Asp Lys Tyr Glu Pro Phe
Trp Glu Asp Glu Glu Lys Asn Glu Ser 50 55 60 Gly Leu Thr Glu Tyr
Arg Leu Val Ser Ile Asn Lys Ser Ser Pro Leu 65 70 75 80 Gln Lys Gln
Leu Pro Ala Phe Ile Ser Glu Asp Ala Ser Gly Tyr Leu 85 90 95 Thr
Ser Ser Trp Leu Thr Leu Phe Val Pro Ser Val Tyr Thr Gly Val 100 105
110 Phe Val Val Ser Leu Pro Leu Asn Ile Met Ala Ile Val Val Phe Ile
115 120 125 Leu Lys Met Lys Val Lys Lys Pro Ala Val Val Tyr Met Leu
His Leu 130 135 140 Ala Thr Ala Asp Val Leu Phe Val Ser Val Leu Pro
Phe Lys Ile Ser 145 150 155 160 Tyr Tyr Phe Ser Gly Ser Asp Trp Gln
Phe Gly Ser Glu Leu Cys Arg 165 170 175 Phe Val Thr Ala Ala Phe Tyr
Cys Asn Met Tyr Ala Ser Ile Leu Leu 180 185 190 Met Thr Val Ile Ser
Ile Asp Arg Phe Leu Ala Val Val Tyr Pro Met 195 200 205 Gln Ser Leu
Ser Trp Arg Thr Leu Gly Arg Ala Ser Phe Thr Cys Leu 210 215 220 Ala
Ile Trp Ala Leu Ala Ile Ala Gly Val Val Pro Leu Val Leu Lys 225 230
235 240 Glu Gln Thr Ile Gln Val Pro Gly Leu Asn Ile Thr Thr Cys His
Asp 245 250 255 Val Leu Asn Glu Thr Leu Leu Glu Gly Tyr Tyr Ala Tyr
Tyr Phe Ser 260 265 270 Ala Phe Ser Ala Val Phe Phe Phe Val Pro Leu
Ile Ile Ser Thr Val 275 280 285 Cys Tyr Val Ser Ile Ile Arg Cys Leu
Ser Ser Ser Ala Val Ala Asn 290 295 300 Arg Ser Lys Lys Ser Arg Ala
Leu Phe Leu Ser Ala Ala Val Phe Cys 305 310 315 320 Ile Phe Ile Ile
Cys Phe Gly Pro Thr Asn Val Leu Leu Ile Ala His 325 330 335 Tyr Ser
Phe Leu Ser His Thr Ser Thr Thr Glu Ala Ala Tyr Phe Ala 340 345 350
Tyr Leu Leu Cys Val Cys Val Ser Ser Ile Ser Ser Cys Ile Asp Pro 355
360 365 Leu Ile Tyr Tyr Tyr Ala Ser Ser Glu Cys Gln Arg Tyr Val Tyr
Ser 370 375 380 Ile Leu Cys Cys Lys Glu Ser Ser Asp Pro Ser Ser Tyr
Asn Ser Ser 385 390 395 400 Gly Gln Leu Met Ala Ser Lys Met Asp Thr
Cys Ser Ser Asn Leu Asn 405 410 415 Asn Ser Ile Tyr Lys Lys Leu Leu
Thr 420 425
* * * * *
References