U.S. patent application number 12/079441 was filed with the patent office on 2008-07-31 for neuronal serine threonine protein kinase.
Invention is credited to Gisela Eisenhardt, Achim Fischer, Bernhard Gotz, Birgitta Kammandel, Bettina Klaussner, Rohini Kuner, Stephanie Jomana Naim, Dieter Newrzella, Morltz Rossner, Armin Schneider, Markus Schwaninger, Annette Trutzel.
Application Number | 20080182314 12/079441 |
Document ID | / |
Family ID | 7642396 |
Filed Date | 2008-07-31 |
United States Patent
Application |
20080182314 |
Kind Code |
A1 |
Schneider; Armin ; et
al. |
July 31, 2008 |
Neuronal serine threonine protein kinase
Abstract
The present invention relates to a gene and the coded protein
thereof that is involved in the development of sequelae of local
ischaemia. The new protein is a serine threonine protein kinase and
provides a new therapeutic approach to the prophylaxis and therapy
of apoplexy.
Inventors: |
Schneider; Armin;
(Heidelberg, DE) ; Klaussner; Bettina; (Auckland,
NZ) ; Fischer; Achim; (Heidelberg, DE) ;
Newrzella; Dieter; (Dossenheim, DE) ; Gotz;
Bernhard; (Heidelberg, DE) ; Rossner; Morltz;
(Schwetzingen, DE) ; Eisenhardt; Gisela;
(Heidelberg, DE) ; Kuner; Rohini; (Heidelberg,
DE) ; Trutzel; Annette; (Frankenthal, DE) ;
Kammandel; Birgitta; (Heidelberg, DE) ; Naim;
Stephanie Jomana; (Heidelberg, DE) ; Schwaninger;
Markus; (Heidelberg, DE) |
Correspondence
Address: |
NEEDLE & ROSENBERG, P.C.
SUITE 1000, 999 PEACHTREE STREET
ATLANTA
GA
30309-3915
US
|
Family ID: |
7642396 |
Appl. No.: |
12/079441 |
Filed: |
March 27, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10276645 |
Jun 4, 2003 |
7361502 |
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PCT/EP01/05660 |
May 17, 2001 |
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12079441 |
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Current U.S.
Class: |
435/194 ;
530/387.1; 536/23.2 |
Current CPC
Class: |
A61P 1/04 20180101; A61P
21/00 20180101; A61P 25/16 20180101; A01K 2217/05 20130101; A61P
35/02 20180101; A61P 19/02 20180101; A61K 51/0491 20130101; A61P
37/06 20180101; A61P 25/00 20180101; A61P 35/00 20180101; A61K
38/00 20130101; A61P 31/12 20180101; C12N 9/1205 20130101; A61P
25/08 20180101; A61P 43/00 20180101; A61P 25/14 20180101; A61P
31/18 20180101; A61P 37/08 20180101 |
Class at
Publication: |
435/194 ;
536/23.2; 530/387.1 |
International
Class: |
C12N 9/12 20060101
C12N009/12; C07H 21/04 20060101 C07H021/04; C07K 16/00 20060101
C07K016/00 |
Foreign Application Data
Date |
Code |
Application Number |
May 17, 2000 |
DE |
100 24 171.9 |
Claims
1-30. (canceled)
31. An isolated nucleic acid that codes for a neuronal serine
threonine protein kinase, selected from the group consisting of: a)
a nucleic acid comprising SEQ ID NO: 3; b) a nucleic acid that
codes for a protein comprising SEQ ID NO: 7; and c) a nucleic acid
that codes for a protein having serine threonine kinase activity,
said protein exhibits at least 90% identity to a protein comprising
SEQ ID NO: 7.
32. The nucleic acid according to claim 1 that codes for a protein
comprising SEQ ID NO: 7.
33. The nucleic acid according to claim 1, wherein the nucleic acid
is a DNA.
34. A nucleic acid construct comprising an isolated nucleic acid
selected from the group consisting of: a) a nucleic acid comprising
SEQ ID NO: 3; b) a nucleic acid that codes for a protein comprising
SEQ ID NO: 7; and c) a nucleic acid that codes for a protein having
serine threonine kinase activity, said protein exhibits at least
90% identity to a protein comprising SEQ ID NO: 7.
35. A nucleic acid construct according to claim 4, wherein the
construct is present in a plasmid.
36. An isolated protein selected from the group consisting of: a) a
protein comprising SEQ ID NO: 7; b) a protein having serine
threonine kinase activity, wherein said protein exhibits at least
90% identity to a protein comprising SEQ ID NO: 7.
37. The protein according to claim 6, wherein said protein
comprises SEQ ID NO:7.
38. An antibody that specifically targets the protein according to
claim 6.
39. An isolated protein selected from the group consisting of: a) a
protein comprising SEQ ID NO: 8; and b) a protein having serine
threonine kinase activity, wherein said protein exhibits at least
90% identity to a protein comprising SEQ ID NO: 8.
40. The protein according to claim 9, wherein said protein
comprises SEQ D NO:8.
41. An antibody that specifically targets the protein according to
claim 9.
Description
[0001] The present invention relates to a serine threonine protein
kinase, nucleic acids that code this kinase and their use in the
diagnosis and therapy of neuronal and neoplastic diseases.
[0002] Apoplexy, also known as stroke, cerebral apoplexy and
apoplectic shock, is involved in about 15% of all deaths, with men
and women being affected equally. Symptoms range from disturbances
of consciousness to coma, and often also encompasses spastic
hemiplegia, the most disparate symptoms of central-motor and
sensory loss as well as focal or generalised convulsions. Every
instance of apoplexy involves a circulatory disorder in the
localised cerebral region that is associated with oxygen
deficiency. Two basic mechanisms act as triggers. Firstly, massive
bleeding, encephalorrhagia, which is involved in 15% of cases, with
this generally occurring in striolenticular arteries following
vascular rupture, as a result of which limited cerebral regions are
destroyed. This mechanism results in a high degree of lethality.
The primary disease, which came in question, is in particular
hypertonia, arteriosclerosis, intracranial aneurysm and, more
rarely, consumptive coagulopathy. A cerebral infarction,
encephalomalacia, is involved as the second mechanism, with this
being regarded as the cause in 85% of cases. A necrosis is
generally formed in this connection. Causes for this include
arterial thrombosis, thromboembolism or functional ischaemia
associated with open vascular lumens, e.g. following a drop in
blood pressure. The cerebral infarction "ischaemic necrosis" is the
cause of the apoplexy in about 70-80% of cases. Arteriosclerosis
often represents the underlying causal disease. The rare, slowly
developing symptoms of encephalomalacia are termed "progressive
stroke". Transient symptoms of neurological loss without the
formation of tissue damage ("transient ischaemic attacks") should
be considered as the early signs of a cerebral infarction. A
temporary stenotically induced or microembolism-induced limited
circulatory disturbance is assumed to be the cause. Diagnosis
encompasses not only a general and neurological examination but
also cranial computer tomography, cerebrovascular Doppler
ultrasound examination, spinal tap, dynamic brain scanning, EEG and
nuclear spin resonance tomography.
[0003] The molecular principles of ischaemia and the associated
sequelae are to date virtually unknown. However, it may be assumed
that a complex series of biochemical processes is required until
apoplexy occurs.
[0004] The present invention was intended to address the technical
problem of identifying genes involved in the development of
apoplexy following local oxygen deficiency and thus opening up new
approaches to the prophylaxis and treatment of apoplexy. The
present invention was further intended to address the technical
problem of identifying proteins involved in the development of
apoplexy.
[0005] The said technical problems are solved by a nucleic acid
that codes for a serine threonine protein kinase, with the nucleic
acid being selected from: [0006] a) a nucleic acid with one of the
sequences according to SEQ ID NOs. 1-4 and a nucleic acid that
codes for a protein with a sequence according to one of SEQ ID NOs.
5-8; [0007] b) a nucleic acid that hybridises with a nucleic acid
according to a); [0008] c) a nucleic acid which, taking account of
the degeneration of the genetic code, would hybridise with a
nucleic acid according to a); [0009] d) derivatives of a nucleic
acid according to a)-c) that are obtained by substitution,
addition, inversion and/or deletion of one or more bases; and
[0010] e) a nucleic acid that is complementary to a nucleic acid
according to a)-d).
[0011] For example, in derivatives of the proteins according to SEQ
ID NOs. 5-8, arginine radicals are replaced by lysine radicals,
valine radicals by isoleucine radicals or aspartic acid radicals by
glutamic acid radicals, with the physicochemical properties of the
replaced amino acid and the amino acids to be replaced being very
similar (e.g. spatial filling, alkalinity, hydrophobicity).
However, one or more amino acids may also be replaced within their
sequence, added or removed, or several of these measures may be
combined with one another. The proteins that are thus modified with
respect to SEQ ID NOs. 5-8 have at least 60%, preferably at least
70% and particularly preferably at least 90% sequence identity with
the sequences SEQ ID NOs. 5-8, calculated in accordance with the
algorithm of Altschul et al., J. Mol. Biol., 215, 403-410, 1990.
The isolated protein and its functional variants can be isolated
advantageously from the brain of mammals such as Homo sapiens,
Rattus norvegicus or Mus musculus. The term `functional variants`
should also be understood to mean homologues from other
mammals.
[0012] The nucleic acids according to the invention according to
SEQ ID NOs. 1-4 represent nucleic acids as isolated from the mouse
(SEQ ID NOs. 1 and 2) or humans (SEQ ID NOs. 3 and 4) respectively,
with the nucleic acids according to SEQ ID no. 2 and SEQ ID no. 4
being longer splice variants of SEQ ID no. 1 and SEQ ID no. 3
respectively. Nucleic acids that code for a protein that displays
at least 60%, preferably at least 70% and particularly preferably
at least 90% identity to one of the proteins as coded by SEQ ID
NOs. 1-4 are also regarded as being in accordance with the
invention.
[0013] "Derivatives" of the aforesaid nucleic acids according to
the invention, e.g. allele variants, differ from the said nucleic
acids according to SEQ ID NOs. 1-4 by substitution, addition,
inversion and/or deletion of one or more bases, but with kinase
activity being maintained. Derivatives, such as homologues or
sequentially allied nucleic acid sequences, can be isolated from
all mammalian species, including humans, by current methods via
hybridisation with one of the nucleic acid sequences according to
the invention or fragments thereof.
[0014] The term "functional equivalents" should also be understood
to mean homologues of the nucleic acid according to SEQ ID NOs.
1-4, for example homologues from other mammals, shortened
sequences, single-strand DNA or RNA or PNA of the coding and
non-coding nucleic acid sequence. Functional equivalents of this
kind can be isolated on the basis of the nucleic acids of SEQ ID
NOs. 1-4, for example by standard hybridisation methods or PCR
technology, from other vertebrates, such as mammals.
Oligonucleotides from conserved regions that can be determined by
the expert in the known way are advantageously used for
hybridisation. However, longer fragments of the nucleic acids
according to the invention or the entire sequence may also be used
for hybridisation. Standard conditions for hybridisation vary
according to the nucleic acid used--oligonucleotide, longer
fragment or full sequence--or according to which nucleic acid
type--DNA or RNA--is used for hybridisation. Thus, for example,
melting temperatures for DNA: DNA hybrids are around 10.degree.
lower than those of DNA-RNA hybrids of the same length.
[0015] The term "standard conditions" should, for example depending
on nucleic acid temperatures, be understood to mean between 42 and
58.degree. C. in an aqueous buffer solution with a concentration of
between 0.1-5.times.SSC (1.times.SSC=0.15 M NaCl, 15 mM sodium
citrate, pH 7.2) or additionally in the presence of 50% formamide,
such as for example 42.degree. C. in 5.times.SSC, 50% formamide.
Advantageously, hybridisation conditions for DNA:DNA hybrids are
0.1.times.SSC and temperatures between around 20.degree.
C.-45.degree. C., preferably between around 30.degree.
C.-45.degree. C. For DNA:RNA hybrids, hybridisation conditions are
advantageously 0.1.times.SSC and temperatures between around
30.degree. C.-55.degree. C., preferably between around 45.degree.
C.-55.degree. C. These specified temperatures for hybridisation are
for example calculated melting temperature values for a nucleic
acid with a length of approx. 100 nucleotides and a G+C content of
50% in the absence of formamide. The experimental conditions for
DNA hybridisation are described in relevant genetics textbooks such
as, for example, Sambrook et al., "Molecular Cloning", Cold Spring
Harbor Laboratory, 1989, and can be calculated by formulae familiar
to the expert, for example depending on the length of the nucleic
acids, the nature of the hybrids or the G+C content. Additional
information on hybridisation can be obtained by the expert from the
following textbooks: Ausubel et al. (eds), 1985, Current Protocols
in Molecular Biology, John Wiley & Sons, New York; Hames and
Higgins (eds), 1985, Nucleic Acids Hybridization: A Practical
Approach, IRL Press at Oxford University Press, Oxford; Brown (ed),
1991, Essential Molecular Biology: A Practical Approach, IRL Press
at Oxford University Press, Oxford.
[0016] The term "derivatives" of the sequences according to the
invention according to SEQ ID NOs. 1-4 should also be understood to
mean promoter variants upstream of the coding regions of the
sequences according to the invention; these may be modified by one
or more nucleotide replacements via insertion, addition and/or
deletion without the promoter properties, particularly promoter
strength and inducibility, being impaired. However, the term
"derivative" also covers promoter variants which, based on the
sequences according to the invention, are strengthened in their
activity by nucleotide replacement(s).
[0017] "Neoplastic diseases" are those to do with abnormal growth
behaviour of cells, the loss of intercellular inhibiting
mechanisms, etc. These include, for example, carcinomas as abnormal
proliferations of endodermal cells, lymphoneoplastic diseases,
melanomas, etc.
[0018] A preferred nucleic acid codes for a protein with a sequence
according to one of SEQ ID NOs. 5-8 or a protein that displays at
least 60% identity to one of the said sequences.
[0019] In a further preferred embodiment, the nucleic acid is at
least 60% identical to the coding sections of one of the sequences
according to one of SEQ ID NOs. 1-4.
[0020] In a further preferred embodiment, the nucleic acid codes
for a protein sequence according to one of SEQ ID NOs. 5-8, with a
nucleic acid that codes for SEQ ID no. 7 being particularly
preferred.
[0021] The nucleic acid according to the invention is preferably a
DNA; however, RNA or PNA are also considered.
[0022] As fragments of a nucleic acid according to the invention,
those suitable for inhibiting the expression of a serine threonine
protein kinase in the antisense orientation to a promoter following
incorporation in a host cell are preferred in particular. Such
fragments are preferably at least 10 nucleotides, preferably at
least 50 nucleotides, particularly preferably at least 200
nucleotides long.
[0023] Constructs according to the invention contain the nucleic
acid sequence according to the invention or a fragment thereof in
combination with other sequences, with which they are usually not
associated in the genome of a host cell. Such "foreign sequences"
are preferably genetic control elements, transcription and
translation signals (also termed "expression-controlling elements"
or sequences derived from vectors). The sequences according to the
invention are functionally associated with these elements.
[0024] This association may, depending on the desired application,
lead to an increase or reduction in gene expression. Host organisms
can be transformed with the recombinant nucleic acid constructs
thus produced. In addition to these control sequences, the natural
control of these sequences of the actual structure genes may still
be present and, where appropriate, have been genetically modified
so that natural control has been eliminated and expression of the
genes increased. The gene construct may, however, also be
constructed more simply, i.e. no additional control signals are
inserted upstream of the sequences and the natural promoter with
its control is not removed. The natural control sequences may
instead be mutated in such a way that no further control takes
place and gene expression is increased. Additional advantageous
control elements may also be inserted at the 3' terminal of the
nucleic acid sequences according to the invention. The nucleic acid
sequences according to SEQ ID NOs. 1-4 and/or sequences according
to SEQ ID NOs. 5-8 that code for the corresponding proteins may be
present in one or more copies in the gene construct, or be located
on separate gene constructs. Advantageous control sequences are
present for example in promoters such as cos, tac, trp, tet,
trp-tet, lpp, lac, lpp-lac, lacIq, T7, T5, T3, gal, trc, ara, SP6,
I--PR or the I-PL promoter, which are preferably used in
gram-negative bacteria. Other advantageous control sequences are
present for example in the gram-positive promoters such as amy and
SPO.sub.2, in yeast promoters such as ADC1, MFa, AC, P-60, CYC1 and
GAPDH or in mammalian promoters such as CaM kinase II, CMV, Nestin,
L7, BDNF, NF, MBP, NSE, beta-globin, GFAP, GAP43, tyrosine
hydroxylase, kainate receptor subunit 1 and glutamate receptor
subunit B. In principle, all natural promoters with their control
sequences as referred to above may be used. In addition, synthetic
promoters can be used advantageously. These control sequences are
intended to allow for targeted expression of the nucleic acid
sequences and protein expression. This may, for example, depending
on the host organism, mean that the gene is expressed or
overexpressed only after induction, or that it is expressed and/or
overexpressed immediately. The control sequences or factors may in
this connection preferably positively influence and thereby
increase expression. Strengthening of the control elements may thus
advantageously take place at transcription level by strong
transcription signals being used as promoters and/or "enhancers".
In addition, however, strengthening of translation is also possible
by, for example, stability of the mRNA being improved. The term
"enhancers" should be understood to mean for example DNA sequences
that bring about increased expression via improved interaction
between RNA polymerase and DNA. Other control sequences that can be
cited include, for example, the "locus control regions",
"silencers" or any sub-sequences thereof. These sequences may be
advantageously used for tissue-specific expression. A preferred
embodiment is the combination of the nucleic acid sequence
according to the invention with a promoter, with the promoter 5'
being located "upstream". Other control signals such as
3'-positioned terminators or polyadenylisation signals or enhancers
may be functionally used in the nucleic acid construct. The term
should also be understood to mean complete vector constructs. These
vector constructs or vectors are used for expression purposes in a
suitable host organism. The nucleic acids according to the
invention and/or the genes for the Ser/Thr protein kinase are
advantageously inserted in a host-specific vector that allows for
optimal expression of the genes in the chosen host. Vectors are
well known to the expert and can for example be gleaned from the
book Cloning Vectors (Eds. Pouwels P. H. et al. Elsevier,
Amsterdam-New York-Oxford, 1985, ISBN 0 444 904018). The term
`vectors` should, besides plasmids, also be understood to mean all
other vectors known to the expert such as phages, viruses such as
SV40, CMV, Baculovirus, adenovirus, Sindbis virus, transposons, IS
elements, phasmids, phagemids, cosmids, linear or circular DNA.
These vectors may be replicated autonomically in the host organism
or chromosomally replicated. For integration in mammals, linear DNA
is advantageously used. Expression of the nucleic acids sequences
according to the invention or of the recombinant nucleic acid
construct may be advantageously increased by increasing the number
of gene copies and/or by strengthening control factors that
positively influence gene expression. Thus, a strengthening of
control elements may preferably take place at transcription level
through the use of stronger transcription signals such as promoters
and enhancers. In addition, however, strengthening of translation
is possible by, for example, improving the stability of mRNA or
increasing the scanning efficiency of this mRNA against ribosomes.
To increase the number of gene copies, the nucleic acid sequences
or homologous genes may be incorporated for example in a nucleic
acid fragment or in a vector that preferably contains the control
gene sequences assigned to the respective genes or promoter
activity with an analogous effect. In particular, control sequences
that strengthen gene expression are used. The nucleic acid
sequences according to the invention may be cloned together with
the sequences that code for interacting proteins in an individual
vector and then expressed in the desired organism. Alternatively,
each of the potentially interacting nucleic acid sequences and the
sequences that code for m30 may also be placed in an individual
vector and incorporated separately in the organism in question via
customary methods such as transformation, transfection,
transduction, electroporation or particle guns. In addition, the
nucleic acid construct according to the invention or the nucleic
acids according to the invention may also be expressed in the form
of therapeutically or diagnostically suitable fragments. To
generate the recombinant protein, use may be made of vector systems
or oligonucleotides that extend the nucleic acids or the nucleic
acid construct by specific nucleotide sequences and thus code for
modified polypeptides that serve the purpose of simpler
purification. Hexa-histidine anchors or epitopes that may be
recognised as antigens of various antibodies, for example, are
known as "tags" of this kind in the literature (Studier et al.,
Meth. Enzymol., 185, 1990: 60-89 und Ausubel et al. (eds.) 1998,
Current Protocols in Molecular Biology, John Wiley & Sons, New
York).
[0025] All cells that permit expression of the nucleic acids
according to the invention, their allele variants, their functional
equivalents or derivatives or the recombinant nucleic acid
construct are in principle suitable as host cells. The term `host
cells` should be understood to mean, for example, bacteria, moulds,
yeasts, plant or animal cells. Preferred host cells/organisms are
bacteria such as Escherichia coli, Streptomyces, Bacillus or
Pseudomonas, eukaryotic micro-organisms such as Saccharomyces
cerevisiae, Aspergillus, higher eukaryotic cells from humans or
animals, for example COS, Hela, HEK293, Sf9 or CHO cells. The
combination from the host organism and the vectors appropriate to
the organisms such as plasmids, viruses or phages such as for
example plasmids with the RNA polymerase/promoter system, the
phages 1, Mu or other temperate phages or transposons and/or other
advantageous control sequences form an expression system. The term
`expression systems` should preferably be understood to mean for
example the combination of mammalian cells such as CHO cells and
vectors such as pcDNA3neo vector or HEK293 cells and CMV vector
that are suitable for mammalian cells. Cell-free, in vitro
expression systems are, however, also considered.
[0026] Mammalian tissue, mammalian organs or transgenic mammals are
also considered as hosts according to the invention for expression
of the nucleic acids according to the invention. The said hosts
preferably differ from the wild type in that, compared with the
wild type, they contain a modified quantity of the protein
according to the invention or else a new protein variant of the
protein kinase according to the invention. However, host organisms
in which the naturally occurring nucleic acid that codes for a
protein according to the invention has been either completely or
partially removed or modified in such way that it is
transcription-inactive are also covered.
[0027] The said organisms preferably contain the nucleic acid
according to the invention or the fragment according to the
invention or the construct according to the invention integrated in
a position in the genome that does not match its natural position
as found in the wild type.
[0028] Mice, rats, sheep, cattle or pigs are preferably considered
as transgenic mammals, although non-mammalian organisms such as
plants are also considered as recipients of the sequences according
to the invention. Transgenic organisms may also be what are known
as knock-out animals. Transgenic animals may in this connection
contain a functional or non-functional nucleic acid sequence
according to the invention or a functional or non-functional
nucleic acid construct. A further arrangement according to the
invention for the transgenic animals described above is transgenic
animals in whose germ cells or all or part of the somatic cells or
in whose germ cells and all or part of the somatic cells the
nucleotide sequence according to the invention has been modified by
genetic engineering methods or interrupted by the introduction of
DNA elements. Another possibility for use of the nucleotide
sequence or parts of it is the production of transgenic or
knock-out or conditional or region-specific knock-out animals or
specific mutations in genetically modified animals (Ausubel et al.
(eds.) 1998, Current Protocols in Molecular Biology, John Wiley
& Sons, New York and Torres et al., (eds.) 1997, Laboratory
protocols for conditional gene targeting, Oxford University Press,
Oxford). Via transgenic overexpression or genetic mutation (zero
mutation or specific deletions, insertions or modifications) by
homologous recombination in embryonic stem cells, animal models can
be produced that can supply additional information on the
pathogenesis of apoplexy. Animal models thus produced may represent
essential test systems for evaluating novel therapeutic agents.
[0029] The proteins according to the invention can be obtained by
expression of one of the nucleic acids according to the invention
in a suitable expression system, with expression systems comprising
intact cells preferably being considered. The protein according to
the invention is preferably a protein selected from a protein with
one of the sequences according to SEQ ID NOs. 5-8 or a protein that
is at least 60%, preferably at least 70%, particularly preferably
at least 90% identical to the said sequences, with the "% identity"
with the sequences according to SEQ ID NOs. 5-8 being calculated in
accordance with the algorithm of Altschul et al., J. Mol. Biol,
250, 403-410, 1990. The protein according to the invention and
functional variants thereof may, however, also be isolated from the
brain of mammals such as Homo sapiens, Rattus norvegicus or Mus
musculus. Proteins according to the invention are also those that
can be derived from a protein according to one of SEQ ID NOs. 5-8
by amino acid exchange, with protein kinase activity remaining
essentially unchanged. For example, amino acids in the starting
protein according to SEQ ID NOs. 5-8 may be replaced by those with
similar physicochemical properties (spatial filling, alkalinity,
hydrophobicity, etc.). For example, arginine radicals may be
replaced by lysine radicals, valine radicals by isoleucine radicals
or aspartic acid radicals by glutamic acid radicals. However, one
or more amino acids may also be transposed in their sequence, added
or removed, or several of the said mechanisms may be combined with
one another. The measures for modifying a specified amino acid
sequence to a desired sequence are familiar to the expert. The
production of the antibodies according to the invention that react
with a protein according to the invention is familiar to the
expert. For this purpose, he may for example fall back on the
production of polyclonal antisera or even hybridoma technology for
the production of monoclonal antibodies.
[0030] Inhibitors according to the invention are low-molecular or
even protein-like substances that can selectively inhibit or
completely eliminate the protein kinase activity of the protein
according to the invention. The identification and production of
suitable inhibitors is quite possible for the expert via the use of
conventional protein kinase assays for screening substances.
Suitable screening methods and protein kinase assays are described
below. Suitable substances with desired binding affinity can also
be identified through the use of computer-assisted drug development
(CAD) (cf. for example Bohm, Klebe, Kubinyi, 1996, Wirkstoffdesign,
Spektrum-Verlag, Heidelberg). The inhibitors of protein kinase
according to the invention thus identified are for example suitable
for the prophylaxis and/or therapy of stroke and other neurological
(particularly neurodegenerative) or neoplastic diseases. In the
case of the said low-molecular substances, peptides and proteins
that can enter into a specific interaction with the protein kinase
inhibitor according to the invention are also considered as
inhibitors. Such peptide or protein inhibitors can be identified
for example with the aid of the two-hybrid system or even other
assays. These assays permit the delimitation of amino acids that
are responsible for a specific interaction with other interaction
partners. Furthermore, the protein according to the invention and
its protein kinase activity can be simply tested in a test system
in which the activity of the protein is measured in the presence of
the substance to be tested. Simple measurement methods
(calorimetric, luminometric, fluorescence-based or radioactive
techniques) that permit rapid measurement of a multitude of test
substances are preferably involved (cf. Bohm, Klebe, Kubinyi, 1996,
Wirkstoffdesign, Spektrum-Verlag, Heidelberg). The test systems
described allow the searching of chemical libraries for substances
that have inhibitory or even activating effects on proteins
according to the invention. The signal transduction chain that is
induced in ischaemia and proceeds via the protein according to the
invention can be inhibited with these inhibitors. This allows for
the inhibition or prevention of ischaemic sequelae.
[0031] The intracellular physiological interaction partners of the
protein kinase according to the invention, such as phosphorylation
substrate, and kinase activity-controlling intracellular
interaction partners can be identified via the two-hybrid selection
system mentioned above.
[0032] The protein quantity and also the activity (e.g. specific
phosphorylations) of the proteins with the sequences of SEQ ID NOs.
5-8 can be determined with the aid of antibodies. A further object
of the invention is therefore a method for quantifying the protein
activity of a protein with one of the sequences SEQ ID NOs. 5-8.
Based on the amino acid sequences according to SEQ ID NOs. 5-8,
synthetic peptides can be generated that are used as antigens for
the production of antibodies. It is also possible to use the
polypeptide or fragments thereof for the generation of antibodies.
The term "antibodies" should be understood to mean polyclonal,
monoclonal, human or humanised or recombinant antibodies or
fragments thereof, single chain antibodies or even synthetic
antibodies. The term "antibodies according to the invention or
fragments thereof" should in principle be understood to mean all
immunoglobulin classes such as IgM, IgG, IgD, IgE, IgA or their
subclasses such as the subclasses of IgG or their mixtures. IgG and
its subclasses such as for example IgG1, IgG2, IgG2a, IgG2b, IgG3
or IgGM are preferred. The IgG subtypes IgG1/k or IgG2b/k are
particularly preferred. All shortened or modified antibody
fragments with one or two binding sites complementary to the
antigen, such as antibody parts with a binding site formed from
light and heavy chain corresponding to the antibodies such as Fv,
Fab or F(ab')2 fragments or single-strand fragments, may be cited
as fragments. Shortened double-strand fragments such as Fv, Fab or
F(ab')2 are preferred. These fragments may for example be obtained
enzymatically by cleaving the Fc part of the antibodies with
enzymes such as papain or pepsin, by chemical oxidation or by
genetic manipulation of the antibody genes. Genetically manipulated
unshortened fragments may also be advantageously used. The
antibodies or fragments may be used alone or in mixtures. The
antibody genes for the genetic manipulations can be isolated in the
manner familiar to the expert, for example from hybridoma cells
(Harlow, E. and Lane, D. 1988, Antibodies: A Laboratory Manual,
Cold Spring Harbor Press, N.Y.; Ausubel et al., (eds), 1998,
Current Protocols in Molecular Biology, John Wiley & Sons, New
York). To this end, antibody-producing cells are drawn in and the
mRNA isolated from the cells in the known way, with adequate
optical density of the cells, via cell lysis with guanidine
thiocyanate, acidification with sodium acetate, extraction with
phenol, chloroform/isoamyl alcohol, precipitation with isopropanol
and washing with ethanol. cDNA from the mRNA is then synthesised
with the aid of reverse transcriptase. The synthesised cDNA may be
inserted directly or following genetic manipulation, for example by
"site directed mutagenesis", introduction of insertions,
inversions, deletions or base exchanges into suitable animal,
fungal, bacterial or viral vectors and expressed into the
corresponding host organisms. Bacterial or yeast vectors such as
pBR322, pUC18/19, pACYC184, lambda or yeast mu vectors are
preferred for cloning of the genes and expression in bacteria such
as E. coli or in yeast such as Saccharomyces cerevisiae. Specific
antibodies to the proteins according to the invention may be
suitable both as diagnostic reagents and as therapeutic agents for
diseases in which the protein according to the invention is of
pathophysiological importance.
[0033] The diagnostic kit according to the invention contains one
of the nucleic acids according to the invention, a fragment thereof
or a construct containing this, a protein according to the
invention and/or an antibody specific to the protein according to
the invention, and also the other reagents that usually form part
of diagnostic kits. These include suitable buffer solutions and
other detection reagents.
[0034] With the method according to the invention for the diagnosis
of a risk of apoplexy or assessment of the course of a cerebral
infarction, the patient sample is brought into contact with a
nucleic acid according to the invention and the nucleic acid
hybridising therewith in the patient sample is determined. An
elevated level of nucleic acid that hybridises with the nucleic
acid according to the invention is an indicator of an increased
risk of occurrence of apoplexy. As an alternative to the detection
of nucleic acid in the patient sample, the quantity of protein
according to the invention can also be determined as an indicator
of a risk of apoplexy. The antibodies according to the invention,
for example, may be used for protein detection. An elevated protein
level in the patient sample investigated is also an indicator of an
increased risk of apoplexy. These assays can also be simply
performed quantitatively, with the negative control representing
material from a healthy patient.
[0035] Furthermore, the nucleic acids according to the invention
and protein coded therefrom or oligonucleotides and peptides
thereof and antibodies targeted at them may be used for the
diagnosis of other diseases, particularly neurological or
cardiovascular or immunological or tumour diseases. These materials
may further be used for the diagnosis of genetic predispositions to
specific neurological, neoplastic, cardiovascular and immunological
diseases. In addition, monitoring of treatment of the said diseases
can be performed with these materials.
[0036] A method for the qualitative and quantitative detection of a
nucleic acid according to the invention in a biological sample
comprises the following steps: a) incubation of a biological sample
with a known quantity of nucleic acid according to the invention or
a known quantity of oligonucleotides that are suitable as primer
for amplification of the nucleic acid according to the invention,
b) detection of the nucleic acid according to the invention by
specific hybridisation or PCR amplification, c) comparison of the
quantity of hybridising nucleic acid or of nucleic acid obtained by
PCR amplification with a quantitative standard. A method for the
qualitative and quantitative detection of a protein heteromer
according to the invention or a protein according to the invention
in a biological sample comprises the following steps: a) incubation
of a biological sample with an antibody specifically targeted at
the protein heteromer or at the protein according to the invention,
b) detection of the antibody/antigen complex, c) comparison of the
quantities of the antibody/antigen complex with a quantitative
standard. A biological sample from a healthy organism is usually
removed as standard. In particular, the property, as set out below,
of nucleic acid according to SEQ ID NOs. 1-4 being up-regulated in
accordance with specific pathophysiological stimuli, such as for
example cerebral ischaemias, can be used here. This concerns for
example assessment of the course of diseases (such as stroke),
assessment of therapeutic success, and graduation of the severity
of a disease.
[0037] The pharmaceutical composition according to the invention
contains a nucleic acid according to the invention, a fragment
thereof, a construct containing this, a host cell containing the
said items, a protein according to the invention, an antibody
targeted at it and/or an inhibitor according to the invention,
where appropriate together with the customary excipients and
carriers.
[0038] Therapeutic applications of the items according to the
invention concern the modulation of processes connected with the
phosphorylation of endogenous proteins. These include the following
physiological or pathophysiological processes: influencing of
immunological activation processes (e.g. activation of monocytes, T
cells); influencing of cell death processes, e.g. cascades that
lead to cell death, or of processes that lead to uninhibited
growth. The treatable cell types include in particular neural
cells, tumour cells and cells of the immune system. Lastly, cell
interactions in which protein kinases are involved, e.g. cell
division, cell differentiation, plasticity and regeneration, can be
influenced with the materials according to the invention.
[0039] The nucleic acid according to the invention or fragments
thereof and the construct containing this, the corresponding host
cells and the protein according to the invention, the antibody
according to the invention and/or the inhibitor according to the
invention may in particular be used for the prophylaxis and/or
therapy of neurological, particularly neurodegenerative diseases.
These include in particular stroke, multiple sclerosis, Parkinson's
disease, amyotrophic lateral sclerosis, heterodegenerative ataxias,
Huntington's disease, neuropathies and epilepsies.
[0040] Tumour diseases may also be preferentially diagnosed/treated
therewith. Examples of such tumour diseases include carcinomas of
the colon, rhabdomyosarcoma and bronchial carcinomas.
[0041] Immunological diseases, including autoimmune diseases,
atopies and/or HIV infections and coinfections, may also be
diagnosed/treated by other immunotropic viruses and/or acute and/or
chronic lymphatic leukaemia, and/or acute and/or chronic myeloid
leukaemia and/or primary chronic polyarthritis and/or Crohn's
disease and/or Colitis ulcerosa with the protein kinase according
to the invention.
[0042] The nucleic acids according to the invention can also be
used as part of gene therapy in mammals and in particular in
humans. In gene therapy, the sequences according to the invention
can be introduced either into the body or parts thereof or the
expression of endogenous substances can be regulated, as for
example by means of antisense technology. Oligonucleotides, e.g.
with antisense orientation or hybrid RNA-DNA oligonucleotides that
contain fragments of the sequences according to the invention may
be used for this purpose. In addition, viral constructs containing
a sequence according to the invention may be used. Lastly, bare DNA
containing a nucleic acid according to the invention or parts
thereof may be used.
[0043] The SNPs identified in the nucleic acid according to the
invention are also useful for diagnosis in research into human
hereditary diseases. The nucleic acids according to the invention
may be used to isolate and characterise additional genes for
homologous mRNAs in the murine and human genome with current
methods by homology screening and correlate them with already known
markers for human hereditary diseases. This permits the
identification of additional genes as the cause of specific
hereditary diseases.
DESCRIPTION OF FIGURES
[0044] FIG. 1 shows the principle of the Restriction-Mediated
Differential Display (RMDD) method.
[0045] FIG. 2 Tissue distribution of the protein according to the
invention in the rat: The upper illustration shows a Northern Blot
obtained with a mouse probe from the 3' region of SEQ ID no. 1. The
lower illustration shows the ethidium bromide staining of the RNA
gel shown in the upper part. A single band of the sample is
detected. By far the greatest expression of the protein according
to the invention is evident in the brain; clear transcripts are
also still present in testis and lungs, while the other tissues
display only very weak bands.
[0046] FIG. 3 shows the tissue distribution of the protein
according to the invention in humans.
[0047] This shows quantitative PCR with the aid of the LightCycler
system and use of MTC (multiple tissue cDNA) kits from the company
Clontech. The strongest expression is located in the brain and
lungs and the intestinal organs.
[0048] FIG. 4 shows the induction of the protein according to the
invention by focal cerebral ischaemia.
[0049] This shows quantification of 9B5 mRNA with the aid of the
LightCycler system following induction of focal cerebral ischaemia
in mice. The nucleic acid according to the invention is
up-regulated for two hours after an ischaemic episode. Six hours
after ischaemia, this induction is no longer detectable.
[0050] FIG. 5 shows the induction of the protein according to the
invention by focal cerebral ischaemia (in situ hybridisation of
murine brain)
[0051] This shows in situ hybridisation of 9B5 following induction
of focal cerebral ischaemia in mice. The nucleic acid according to
the invention is up-regulated for two hours after an ischaemic
episode (left=ischaemic=induced side). It is also evident that 9B5
is expressed in neurons. Specific induction is to be found in the
cortex and in the hippocampal region.
[0052] FIG. 6 shows the catalytic region of SEQ ID no. 7, where the
symbols have the following meaning: I=phosphate anchor,
VIb=catalytic region, VII-VIII=activation loop, V-XI=catalytic
domain with subdomains (after Hanks und Hunter, 1995, FASEB J., 9,
576-596); *=activation site, A=ATP binding site; C=active
centre.
[0053] FIG. 7 shows the physiological tree for homologous protein
kinases. H9B5 is the phylogenetically youngest protein and the most
closely related to P78/c-TAK 1. The early cleaving of the C.
elegans protein Par-1 is also clearly shown.
[0054] FIG. 8 shows the domain structure of 9B5.
[0055] FIG. 9 is a hydrophobicity blot according to
Kyte-Doolittle.
[0056] FIG. 10 shows the genomic structure of 9B5 in humans.
[0057] This shows the 18 exons of the 9B5 gene. Exon 16 is subject
to differential splicing. The variant 9B5 is formed without exon
16; the variant 9B5A contains exon 16.
Protein Sequence of 9B5
[0058] The murine protein sequences (SEQ ID NOs. 5 and 6) are
incomplete at the 5' terminal. In the case of the human sequences
according to SEQ ID NOs. 7 and 8, a full-length clone is involved.
This also arises from alignments with homologous protein
kinases.
[0059] In the case of h9B5 (SEQ ID no. 7), an open reading frame
results that codes for a protein with 752 amino acids (82.5 kD
molecular weight; isoelectric point at pH 9.7). The program PSORT
II yields no significant signal sequences, and does not predict
transmembrane regions. A Kyte-Doolittle hydrophobicity plot (FIG.
9) also shows a high proportion of hydrophilic regions and only one
relatively large hydrophobic region (approx. aa 240-255) which does
not, however, seem sufficiently hydrophobic for a transmembrane
region (index >-3). The reported membrane-associated location of
a number of related proteins (p78/c-TAK1, par 1) is, however,
interesting in this context.
[0060] An open reading frame of 688 amino acids with a molecular
weight of 75.3 kD and an isoelectric point at pH 9.8 results for
the sequence h9B5_b (SEQ ID no. 8). As a result of the insertion of
exon 16, a frame shift takes place that leads to an earlier
translation stop.
Classification of 9B5
[0061] A comparison with known protein kinases permits
classification of the protein according to the invention to the
subgroup "CaMK Group II" according to Hanks
(http://www.sdsc.edu/Kinases/pkr/pk_catalvtic/pk_hanks
seq_align_long.html), (cf. Hanks and Lindberg, Methods Enzymol,
200, 525-32, (1991); Hanks and Hunter, Faseb J, 9, 576-96,
(1995))). Drewes et al. (Drewes, et al., Cell, 89, 297-308, (1997))
have already postulated for MARK1/MARK2 and p78a new subgroup in
the already defined SNF1/AMPK subgroup of the CaMKII group. Based
on alignment of the most highly homologous proteins (9B5, Par1,
cTAK1/p78, MARK1, EMK) and the phylogenetic tree (FIG. 7), the
possibility exists that 9B5 defines a new subgroup. At the
carboxy-terminus, 9B5 deviates sharply from the other protein
kinases. The kinases in this group are characterised by a highly
conserved carboxy-terminus domain that terminates with the sequence
"ELKL". EMK has therefore also been designated as "ELKL motif
kinase". It should be assumed that protein kinases still exist that
are similar to 9B5 in the carboxy-terminus and that can be
identified via homology screens. A search with the program
"tblastn" against the EMBL nucleotide database has to date yielded
no evidence of already known homologues in the carboxy-terminus
region.
[0062] Proteins with the target sequence KXGS and/or KVGS may form
the physiological substrate of the protein according to the
invention. Accordingly, the Tau protein or the proteins MAP1 and 2
may represent a physiological substrate of the protein kinase
according to the invention.
Subdomains of the Catalytic Domain: 9B5 is a Serine Threonine
Protein Kinase
[0063] The important catalytic subdomains can be clearly defined
via comparisons with other protein kinases (see FIG. 6). The
activation sites can likewise be shown (a threonine and a serine
that may be phosphorylated; pos. 211 and 215 in h9B5, FIG. 6). A
mutation of these amino acids, for example to alanine, leads to
constitutive inactivation. Mutation of the lysine radical (ATP
binding) in subdomain II (see FIG. 6) likewise leads to
inactivation. This finding can be utilised for further experiments,
e.g. using dominant negative effects. A mutation of pos. 211 (Ser)
and 215 (Thr) to glutamate or aspartate should lead to constitutive
activation as the negatively charged groups may imitate
phosphorylation (Huang and Erikson, Proc Natl Acad Sci USA, 91,
8960-3, (1994)). These mutants may be exploited for overexpression
of the specific kinase activity in cells or transgenic animals.
[0064] Subdomain I is known to act as a kind of clasp that anchors
the non-transferrable phosphate groups of ATP. In this connection,
the peptide sequence GKGNFAKV in 9B5 fits in well with consensus
motif GXGXXGXV (Hanks and Hunter, Faseb J, 9, 576-96, (1995)). In
subdomain II, chiefly lysine (labelled "A" in FIG. 6) is important;
this anchors and orientates the alpha and beta phosphates of ATP
and is essential for enzyme function. Subdomain VIb is
characterised by the consensus sequence HRDLKXXN. This is also very
well preserved in 9B5: HRDLKAEN.
[0065] Aspartate (D) is in this connection probably the proton
acceptor for the attacking hydroxyl group during the
phosphotransfer reaction (Hanks and Hunter, Faseb J, 9, 576-96,
(1995)). Subdomain VII forms with Mg2+ ions a chelate complex that
encloses the gamma-phosphate and thus orientates this group for the
transfer. The sequence DFG is practically invariable. The preserved
sequence APE in subdomain VIII is similarly maintained. This domain
is responsible for peptide recognition. Inhibitor peptides can also
bind here. Many protein kinases are activated by phosphorylation
within this subdomain. This domain is fully preserved in EMK, p78
and MARK1. In the case of MARK1, it has already been shown by
direct sequencing that threonine (T) and serine (S) can be
phosphorylated, and MARK1 is thereby activated. Owing to this
preservation, it may be assumed with the utmost probability that
9B5 also phosphorylates at these sites and thus can be controlled.
Autophosphorylation possibly takes place; activation by another
kinase is also conceivable. The tyrosine (Y) in this domain is
possibly also a phosphorylation site (as for example in the case of
Erk1/2). Subdomain IX is also involved in peptide binding
(hydrophobic interaction).
[0066] On account of this structural development, it can be assumed
with certainty that 9B5 is an active protein kinase (serine
threonine kinase).
Tissue Expression of 9B5
[0067] The tissue expression of 9B5 has been investigated in mice
and humans. In mice, a "multiple tissue northern" was performed
with various tissues (FIG. 2). A fragment from the 3' region of the
mouse cDNA was used as sample. Here, expression takes place in the
brain, testicles and lungs.
[0068] In humans, a "multiple tissue northern" (Clontech) was
initially performed with 2 different probes (from the 3' and 5'
regions). However, no clear signal could be obtained, which was
attributed to the low incidence of 9B5 in humans. A quantitative
PCR was therefore performed with the aid of the LightCycler (Roche
Diagnostics, Mannheim). cDNA samples of 8 human tissues already
quantitatively standardised on 4 different housekeeping genes were
used (manufactured by Clontech). Plasmid h9B5-663, which contained
an amplified fragment of human 9B5-cDNA, was used as control. A
touchdown protocol was chosen as PCR program. Amplification of a
product with a melting point of 90.degree. C. took place in all
tissue samples; this coincided with the control PCR. A subsequent
gel analysis of the fragments yielded a product of approx. 660 bp,
i.e. the expected size. 9B5 in humans is actually not very strongly
expressed; amplification becomes visible in the LightCycler only at
around cycle 32. The following primers were used:
seq_h9b5_s1 GTTGCCATCAAGATTATC (in exon 3) seq_h9b5-a4
CATGATTTGCTCGAGAGTAC (in exon 9) Quantitative analysis (FIG. 3)
shows a relatively ubiquitous tissue distribution with higher
concentrations in the brain and lungs, liver, kidneys and
pancreas.
Regulation by Focal Cerebral Ischaemia, a Stroke Model
[0069] The serine threonine kinase 9B5 according to the invention
was identified by a method for the cloning of differentially
regulated genes (RMDD) in the ischaemic hemisphere of mice
following focal cerebral ischaemia. The animal model for focal
cerebral ischaemia represents a valid model for human ischaemic
stroke. To bring about the focal cerebral ischaemia, use was made
of the so-called thread model, in which a coated nylon thread is
passed through the A. carotis interna up to the end of the A.
cerebri media and induces an ischaemic stroke (Clark et al.,
Neurol. Res., 19, 641-648, (1997)). In cerebral ischaemia,
regulation of gene expression plays a crucial role in the course
and extent of neuronal damage (Koistinaho and Hokfelt, Neuroreport,
8, i-viii, (1997, Schneider et al., Nat Med, 5, 554-9, (1999)). In
particular, "immediate early" genes play a role here (Atkins et
al., Stroke, 27, 1682-1687, (1996)), such as cox-2, (Nogawa et al.,
J. Neurosci., 17, 2746-2755, (1997)).
[0070] 9B5 expression was, following focal cerebral ischaemia,
investigated over three timescales: firstly, in a transient
ischaemia following two reperfusion periods (ischaemia for 90 mins,
reperfusion for 2 h and 6 h), and secondly in a permanent ischaemia
of 24 h (FIG. 4). RNA was extracted from the two hemispherical
halves of 3-4 brains without brain stem and cerebellum (Fasttrack
kit, Invitrogen). With the aid of the LightCycler.TM. system (Roche
Diagnostics, Mannheim), a quantitative PCR was performed. The cDNA
content of the samples was standardised to the expression of
cyclophilin and S20 (Schneider et al., Proc Natl Acad Sci USA, 92,
4447-51, (1995)). The primers used for the amplification of
cyclophilin were:
TABLE-US-00001 cyc5 ACCCCACCGTGTTCTTCGAC acyc300
CATTTGCCATGGACAAGATG
and for the amplification of murine 9B5:
9_B5_(1)1s TATGATCGAACCTCCTTCATGCC
[0071] 9_B5_(1)1a ATGTCCAGAACTGGGCCTAGCG (These primers amplify an
amplimer of 556 bp, which is located at the 3' terminal of the
murine cDNA).
[0072] In actual fact, clear up-regulation of 9B5-RNA by a factor
of 7-8 on the ischaemic (left) half of the brain appears 2 h after
the ischaemic event (middle cerebral artery occlusion for 90 mins
and reperfusion for 2 h; (FIG. 4); the error bars show standard
deviations--these arise from measurements with thrice serially
diluted cDNA samples and thus reflect the reliability of the
measurement results). After 24 h (in a permanent model), on the
other hand, no difference was any longer detectable.
[0073] In-situ hybridisation was also performed with a 1.6 kb long
probe from the 3' region of murine 9B5-cDNA (sense and antisense in
each case). The procedure adopted for this was essentially as per
the protocol of the company Roche Diagnostics (digoxigenin system)
following modifications by Rossner et al. (Mol Cell Neurosci, 9,
460-475, (1997)). Clear induction is apparent on the ischaemic side
(left), particularly in neurons of the hippocampal region and
cortex, less so in the thalamus (FIG. 5). It also becomes clear
that 9B5 is predominantly expressed in neurons, and is subject to
control there.
[0074] This shows that 9B5 plays an important role in the
pathogenesis of stroke, with the up-regulation of 9B5 playing a
similar role to that, for example, of the known serine threonine
kinases (e.g. JNK, p38).
[0075] A multitude of serine threonine kinases are involved in cell
death processes (e.g. ASK1 (Tobiume, et al., Biochem Biophys Res
Commun, 239, 905-10, (1997, Berestetskaya, et al., J Biol Chem,
273, 27816-23, (1998, Chen, et al., Oncogene, 18, 173-80, (1999)),
DAP (Inbal, et al., Nature, 390, 180-4, (1997, Levy-Strumpf and
Kimchi, Oncogene, 17, 3331-40, (1998)), DRAKs (Sanjo, et al., J
Biol Chem, 273, 29066-71, (1998)), ZIP (Kawai, et al., Mol Cell
Biol, 18, 1642-51, (1998))), DRP-1 (Inbal, et al., Mol Cell Biol,
20, 1044-54, (2000)).
[0076] This up-regulation is evidence of a new transcriptional
control mechanism for protein kinases, to date the only known
example of this in the mammalian system. Interestingly, the
up-regulation of several MAP kinases was only very recently found
in a systematic study of the yeast transcriptome (Roberts, et al.,
Science, 287, 873-80, (2000)). This might represent a new general
mechanism for the regulation of protein kinases.
Pharmacological Significance of 9B5 in Neurodegenerative,
Neoplastic and Other Diseases
[0077] Approaches to inhibiting/influencing signal transduction
pathways "downstream" of a membrane-based receptor have recently
been gaining in importance in pharmacological research. These
approaches will in future probably play an important part in the
treatment of human diseases, particularly in the case of diseases
that have hitherto been poorly treatable or untreatable (Kletsas
and Papavassiliou, Exp. Opin. Invest. Drugs, 8, 737-746, (1999)).
Advantages of these approaches are that they can first influence
events that can be brought about by several stimuli, and have a
common terminal section; secondly, cellular events temporally
follow the triggering stimulus, and are thus open to intervention
for longer.
[0078] Examples of successful interventions in such signal cascades
include inhibitors for caspases that can block apoptosis processes
for even longer after a triggering stimulus. The transcription
factor NF-kappaB and these activating processes have also attracted
particular attention. For example, cloning of the I-kappaB kinases
was pursued with the aim of finding specific inhibitors for
NF-kappaB-mediated gene transcription. Differential transcription
profiling has recently been gaining in importance in pharmaceutical
research to identify possible new points of departure for drugs.
Transcriptional control, i.e. control of the quantity of mRNA in a
gene in the cell, is an essential stage in the cell's response to
stimuli, in addition to protein phosphorylations, protein
degradation, etc. 9B5 is evidently subject to very rapid control by
transcriptional activation, as shown above. Rapidly controlled
genes of this kind are often involved in critical key positions for
cellular processes.
[0079] A pharmacological influence on 9B5 is possible in the
following way: 1. an effect on the quantity of transcript in the
cell, for example suppression of rapid up-regulation following
pathological processes (e.g. via antisense technology); 2.
inhibition of the enzymatic activity of 9B5, particularly kinase
activity (e.g. via a kinase inhibitor; 3. inhibition of an
interaction with one or more other molecules, e.g. downstream
protein kinases, or adaptors.
[0080] Specific inhibitors for a number of MAP protein kinases have
been developed recently. One example is PD98059, an inhibitor of
MEK1. This prevents the phosphorylation of Tau by stimulation with
beta-amyloid. This has possible significance for the treatment of
Alzheimer's disease.
[0081] The antitumour substance UCN-01 is an inhibitor of cdc25c
phosphorylation (Graves, et al., J Biol Chem, 275, 5600-5,
(2000)).
[0082] Inhibitors of the protein kinase according to the invention
can be simply identified with conventional protein kinase assays
and represent an effective aid in controlling the sequelae of
cerebral ischaemia.
[0083] The experimental data on 9B5 prove its central involvement
in processes associated with neuronal cell death, excitation,
plasticity and neurogenesis. The protein according to the invention
represents an important target molecule for inhibiting or reducing
the sequelae of focal ischaemia. Besides this central role in the
development of apoplexy as a consequence of focal ischaemia, the
protein according to the invention might also be an important
target molecule in the treatment of neoplastic diseases, such as
cancer. Here, too, the protein according to the invention or its
gene might represent the target molecule for anticancer agents. The
gene might likewise play an important role in the diagnosis and
therapy of cardiovascular diseases as a number of shared mechanisms
exist for ischaemically induced diseases.
[0084] In a particularly preferred embodiment according to the
invention, the level or activity of the protein kinase according to
the invention is lowered for the prophylaxis and therapy of stroke.
This may for example take place at the level of expression, by
expression/translation of the corresponding nucleic acids being
inhibited or reduced, or at the level of protein activity, by
protein kinase activity being reduced or inhibited by suitable
inhibitors.
[0085] The nucleic acid according to the invention and the protein
coded therefrom open up new therapeutic approaches. Thus, for
example, the level of endogenous nucleic acid can be influenced by
either directly influencing its transcription or even influencing
its translation to protein according to the invention. For example,
gene therapy approaches are considered for this in which, via
co-suppression or antisense technology, the translation of
endogenous transcripts is lowered. New approaches also present
themselves at the level of protein activity by the protein
according to the invention for example representing the target
molecule of pharmaceutical active substances. Thus, the activity of
protein kinase according to the invention can be modified by
pharmaceutical active substances in order to intervene in a
controlling way in the mechanism of apoplexy. The protein kinase
inhibitors mentioned above represent a class of such pharmaceutical
active substances. In principle, provision of the nucleic acid
according to the invention or the protein according to the
invention thus also opens up entirely new approaches to the
prophylaxis or therapy of stroke.
[0086] The following examples elucidate the invention:
EXAMPLES
Example 1
Molecular cloning of 9B5
Induction of the Thread Model in Mice
[0087] To induce focal cerebral ischaemia in c57/bl6 mice,
3-month-old mice were used. Following induction of an inhalation
anaesthesia (70% N.sub.2O, 30% O.sub.2, 0.8-1% halothane), a 5-0
prolene thread (manufactured by the company Ethicon) coated with
0.1% poly-L-lysine was passed via the A. carotis externa into the
A. carotis interna up to the end of the A. cerebri media. The
correct position of the thread is indicated by a drop in the laser
Doppler signal (Perimed company) to 10-20% of the starting signal.
Following the performance of this operation and, where appropriate,
determination of additional physiological parameters (blood
pressure, pulse, blood gases, blood glucose), the mice wake from
the anaesthesia. After specific occlusion periods, the mice are
again subjected to anaesthesia, and the thread is withdrawn.
Reperfusion of the tissue thereby takes place. Following specific
reperfusion periods, the mice are sacrificed by breaking their
necks, and the brains immediately prepared and frozen in dry ice.
In the present case, no reperfusions were performed, only an
occlusion for 90 mins or 24h.
Preparation of mRNA from the Brains
[0088] The mRNA preparation kit manufactured by Invitrogen
(Fasttrack) was used for this purpose.
Performance of the RMDD Protocol (see also FIG. 1)
[0089] The procedure adopted was essentially as per Pat. no. EP 0
743 367A2; U.S. Pat. No. 5,876,932, with the modification that 2
.mu.g polyA-RNA was used for the first-strand synthesis. Following
performance of first-strand, second-strand synthesis, Mbol
restriction, ligation with adaptors is performed. Two successive
PCR reactions with subsets of primer combinations follow. The PCR
reactions are then loaded onto a denaturing polyacrylamide gel and
blotted on a nylon membrane (manufactured by GATC). The
biotin-labelled bands are visualised with the aid of an ordinary
streptavidin peroxidase reaction. PCR samples of the ischaemic and
contralateral hemisphere were applied together to the gel (24 h
MCAO on the right and left and 90 mins MCAO on the right and left).
Bands of differing intensity in the right or left hemisphere are
cut out, and reamplification of the corresponding PCR product
performed. Amplified products obtained are cloned into TOPO TA
vector pcDNA 2.1 (manufactured by Invitrogen) and sequenced with T7
and M13 rev primers (ABI 3700 capillary electrophoresis
sequencer).
Cloning of 9B5
[0090] During the performance of this method, a sequence was
noticed that seemed to be up-regulated after 90 mins on the
ischaemic side. This was called 9B5. A LightCycler analysis
confirmed rapid regulation after MCAO (90 mins MCAO and 2 h
reperfusion) (FIG. 4).
[0091] The isolated 3'-positioned PCR fragment was used to
hybridise a murine brain bank, in which there were several clones
that contained sequence parts of SEQ ID no. 1 and SEQ ID no. 2. A
mouse sample from the 5' region was used to screen a humane foetal
brain bank in lambdaZapII (Stratagene). In this connection, 2
different sequences also resulted from several clones, which
presumably represent splice variants of the same gene (SEQ ID NOs.
3 and 4), resulted from several clones.
[0092] The detailed production of the human cDNA library used is
set out below:
Production of the Human cDNA Library
[0093] Using the cDNA synthesis kit manufactured by the company
Stratagene, corresponding cDNA libraries were, on the basis of 2 pg
human foetal brain mRNA (manufactured by Clontech) and 5 .mu.g
mRNA, produced from adult murine brain, with the procedure adopted
essentially being in accordance with the manufacturer's details. To
synthesise the first-strand cDNA, an oligodT primer was used in
accordance with the manufacturer's details. The cloning-compatible
(EcoRI/XhoI) double-stranded cDNA fragments were selected by size
(in accordance with manufacturer's details/Stratagene) and ligated
into the plasmid vector pBluescript SKII (Stratagene). The ligation
was transformed by electroporation in E. coli (DH10B, Gibco) and
amplified on LB ampicillin agar plates. The plasmid DNA was
isolated via alkaline lysis and ion exchanger chromatography
(QIAfilter kit, manufactured by Qiagen).
[0094] The complexity of individual clones was 4 million for the
foetal human brain cDNA bank. From each cDNA bank, 24 individual
clones were randomly analysed by insert sizes, which showed a size
distribution of 800 bp to 4.5 kB; the mean length of the cDNA
inserts was for the human bank approx. 1.2 kB.
Example 2
Performance of a Reporter Gene Assay
[0095] The sequence of 9B5 obtained can be used to obtain
information on the arrangement of the protein in signal
transduction cascades. To this end, the open reading frame of the
gene is cloned into a current expression vector (e.g. pCMV-tag,
manufactured by Stratagene). This construct can be transfected with
other constructs together in eukaryotic cells (e.g. by the calcium
phosphate method, see Ausubel et al., Current Protocols in
Molecular Biology, New York, 1997). These may be reporter
constructs, e.g. a luciferase gene under the control of a minimal
promoter with several binding sites for, for example, the
transcription factor NF-kappaB or AP-1. Extracts from the cells may
then be subjected to measurement in the luminometer (e.g.
manufactured by the company Bertold). An increase in the luciferase
value indicates influencing of the signal transduction pathway that
results in the activation of a specific transcription factor.
Combinations with expression constructs for other genes (e.g. MAP
kinases) may provide information on the precise position of 9B5 in
signal cascades. These reporter assays can also be performed with
other systems, e.g. lacZ or chloramphenicol transferase (CAT
assays), without the principle of the assays being influenced.
[0096] In the same way, ready-made kits (e.g. Mercury in vivo
kinase assay kits, manufactured by Clontech) may also be used, with
the Tet repressor being expressed in fusion with the transactivator
domain of a phosphorylation target (transcription factors, e.g.
Jun). Activation of a luciferase construct under the control of a
Tet repressor element only takes place if specific phosphorylation
of the transactivator domain by a kinase (e.g. 9B5) occurs. In this
way, arrangement in a cellular signal transduction pathway is
possible.
Example 3
Kinase Assays
[0097] Protein kinases are biochemically very well characterised.
The kinase activity of 9B5 can be demonstrated by cloning the open
reading frame of 9B5 into an expression vector with an epitope tag
(e.g. pcDNA-myc-his) and transfecting it in eukaryotic cells (e.g.
Cos cells). After 48 h, extracts can be obtained from these cells
and immunoprecipitation performed with a myc-specific antibody and
proteinA beads. In a kinase buffer in the presence of
.gamma.-.sup.32P-ATP, a kinase reaction is performed. The proteins
are then denatured and separated on an SDS-PAGE gel.
Autoradiography is then performed. Labelled bands indicate the
kinase activity of 9B5. Autophosphorylation of the kinase itself
often takes place as well. Indications of possible phosphorylation
targets are also provided by cotransfections with potential
targets, e.g. various MAP kinases that are also provided with a tag
and can be immunoprecipitated.
[0098] Kinase assays may also be performed with 9B5 that has been
transcribed/translated in vitro (e.g. T7 reticulocyte system
manufactured by the company Promega). Protein that has been
expressed in E. coli, e.g. as GST fusion protein or as HIS-tagged
construct, can also be used. These tags may in this connection be
used for purifying the protein. The purified proteins can then be
incubated in kinase buffer with potential substrates. MBP (myelin
basic protein) that is frequently non-specifically phosphorylated
by Ser/Thr kinases is often used here.
[0099] The kinase domain of protein kinases is very well defined.
The mutation of individual amino acids in the phosphate transfer
domain is often sufficient for loss of function of the protein
(e.g. K709M in the case of ASK1 (Chang et al., Science, 281,
1860-3, (1998)); K90A in DRAK 1, K62A in DRAK2 (Sanjo et al., J
Biol Chem, 273, 29066-71, (1998)); KK429-430M in NIK (Sanjo et al.,
J Biol Chem, 273, 29066-71, (1998)); K63W in TAK1 (Ninomiya-Tsuji
et al., Nature, 398, 252-6, (1999))). These inactive kinases are
often of great importance as dominant-negative inhibitors for
evaluation of the cell pathways, e.g. in cotransfection experiments
and kinase assays (Ninomiya-Tsuji et al., Nature, 398, 252-6,
(1999)). Mutation of this kind can also be performed with 9B5.
[0100] The specific phosphorylation of target protein can also be
demonstrated with phosphorylation-specific antibodies, e.g.
Phospho-SerThr/Tyr monoclonal antibody, mouse IgG2b, produced by
the company Clontech.
[0101] Further examples of kinase assays that are commonly used in
the technical arena can, for instance, be gleaned from the book
Protein Phosphorylation, A practical approach, ed. D. G. Hardie,
2nd ed., Oxford, 1999, particularly chapters 9 and 10.
Example 4
Identification of the Phosphorylation Target of 9B5
[0102] The identification of phosphorylation targets of 9B5 may,
for example, take place via interaction screening. Somewhat classic
peptide expression banks in the lambda bacteriophages (the most
used system is the vector lambda-gt11, see Ausubel et al., Current
protocols in molecular biology, New York, 1997) may be used in this
connection. One approach is the cloning of 9B5 into a bacterial
expression vector with the incorporation of a purification tag
(e.g. poly-histidine or GST) and a consensus phosphorylation site
for protein kinase A (sequence RRASV). 9B5 may thus be expressed
and purified in bacteria in accordance with standard methods. 9B5
can in this way be labelled as a scavenger with .sup.33P or
.sup.32P by means of incubation with protein kinaseA. The
expression bank can then be incubated with the labelled 9B5.
Following exposure on autoradiograms, positive clones can be
identified and be isolated and sequenced by standard methods. This
technique can also be adapted in the following way: the
autophosphorylation property of 9B5 can be utilised to label 9B5
with .sup.33P-.gamma.ATP via simple incubation. The method can also
be modified in such a way that the expression bank is incubated
with purified 9B5 and .sup.33P-.gamma.ATP under phosphorylation
conditions (phosphorylation buffer, etc.), and expressed peptides
are actively labelled by 9B5. However, this method is more unstable
than that described above. Sequenced peptides can be used to
formulate a consensus recognition and phosphorylation sequence.
This permits the identification of potential substrates via
bioinformatic methods. Candidates can be verified by expression and
incubation with 9B5. Examples of the successful performance of
these forms of expression screening are contained in (Mochly-Rosen
and Gordon, Faseb J, 1Z 35-42, (1998, Blanar and Rutter, Science,
256, 1014-8, (1992, Chapline et al., J Biol Chem, 268, 6858-61,
(1993, Chapline et al., J Biol Chem, 271, 6417-22, (1996, Kaelin et
al., Cell, 70, 351-64, (1992, Songyang et al., Curr Biol, 4,
973-82, (1994)).
[0103] The phosphorylation target of 9B5 can also be identified in
a yeast-two-hybrid screen (Fields and Song, Nature, 340, 245-6,
(1989)). For example, the interaction of Ras and c-Raf (a Ser/Thr
kinase) was discovered in a y2h system (Fields and Song, Nature,
340, 245-6, (1989)). Interaction of the Ser/Thr kinase SNF1 with
SNF4 is also virtually a prototype for the y2h system (Fields and
Song, Nature, 340, 245-6, (1989)). In principle in an equivalent
way to the yeast screens, mammalian systems can also be used
(Fields and Song, Nature, 340, 245-6, (1989)). In the case of a y2h
screen, the open reading frame of 9B5 is cloned into a so-called
"bait vector" with the GAL4 binding domain (e.g. pGBT10,
manufactured by Clontech). A so-called "prey-library" in a yeast
strain can thus be searched for interacting proteins in accordance
with several current protocols. It can in this connection often be
useful to use kinase-negative mutants as these often interact in a
more stable manner with the phosphorylation target. Serine
threonine kinases in a synthesis pathway may be brought into
spatial proximity by adapter molecules in order to be able to
perform specific phosphorylations better (Chang et al., Science,
281, 1860-3, (1998)), (Yasuda et al., Mol Cell Biol, 19, 7245-54,
(1999, Whitmarsh and Davis, Trends Biochem Sci, 23, 481-5, (1998,
Whitmarsh et al., Science, 281, 1671-4, (1998)). It is therefore
also possible to encounter the phosphorylation targets via two
steps in the yeast-two-hybrid system by first cloning an adaptor
protein and finding the specific target molecule with this as
"bait". All in all, mapping experiments for interaction domains can
also be performed with the y2h system.
[0104] It is also possible to use co-immunoprecipitations from
cells transfected with 9B5 expression vectors to purify proteins
binding to them, and to identify the genes via protein sequencing
methods (e.g. MALDI).
[0105] It is also possible, following immunoprecipitation with a
subsequent kinase assay from a cell extract, to purify the
phosphorylated bands and to sequence these.
[0106] Further examples of the identification of protein kinase
substrates that are commonly used in the technical arena can be
gleaned from, for instance, the book Protein Phosphorylation, A
practical approach, ed. D. G. Hardie, 2nd ed., Oxford, 1999.
Example 5
Apoptosis Assays
[0107] Very many previously identified serine threonine kinases are
involved in apoptotic processes, e.g. ASK1 (Zhang et al., Proc Natl
Acad Sci USA, 96, 8511-5, (1999)), (Ichijo et al., Science, 275,
90-4, (1997)), DRAKs (Zhang et al., Proc Natl Acad Sci U S A, 96,
8511-5, (1999)), (Ichijo et al., Science, 275, 90-4, (1997)). The
involvement of 9B5 in apoptotic cascades can be further
investigated by transfecting expression constructs with 9B5 in
eukaryotic cells, and then investigating the induction of
apoptosis. This may, for example, take place via staining with
annexin (manufactured by Roche Diagnostics), by antibodies that
recognise the active form of caspase-3 (manufactured by New England
Biolabs), or by ELISAs that recognise DNA-histone fragments
(cell-death elisa, Roche Diagnostics). This induction of apoptosis
may be cell type-specific, and so several cell lines and primary
cells must be investigated. The induction of apoptosis may also be
stimulus-specific, and so several stress situations may be helpful
in answering this question, e.g. heat shock, hypoxia conditions,
cytokine treatments (e.g. II-1, II-6, TNF-alpha), H.sub.2O.sub.2
treatment. On cell types, several customary lines, e.g. Cos cells,
HEK cells, PC12 cells, THP-1 cells and primary cells such as, for
example, neurons and astrocytes are considered, as are other
immortalised and primary cell lines, as required.
Example 6
High-Throughput Screening Assays for the Identification of
Inhibitors of 9B5
[0108] 9B5 can be used to find inhibitors of the interaction with
its interaction partners (e.g. adaptor molecules). This can be
performed, for example, with the FRET (frequency resonance energy
transfer) system by 9B5 being expressed in fusion and purified with
GFP (green fluorescent protein) and its interaction partner with
BFP (blue fluorescent protein). In a cell-free system, the
reduction in emission of BFP can then be used as an indicator of
the presence of an inhibitor when searching complex chemical
banks.
[0109] The main aim behind therapeutic exploitation of the protein
kinase detected is, however, firstly to eliminate the protein
kinase function, as this can be performed most easily. Protein
kinases present themselves in principle for the performance of
high-throughput assays for the identification of inhibitors
(small-molecule inhibitors) of the kinase property of the protein
as the enzyme property itself can be readily used as indicator (see
also example 3, kinase assays).
[0110] Simple implementation of an HTS system for 9B5 can be
performed by the filter assay method of Reuter et al. (Reuter et
al., Methods in Enzymology, 255: 245 (1995)), with MBP being used
as non-specific substrate. This is applied to 96-hole plates that
are suitable for ELISA (e.g. FlashPlates, NEN Life Science
Products, or NUNC). Reaction buffer (3.times. kinase reaction
buffer contains: 60 mM HEPES (pH 7.5), 30 mM MgAc, 0.15 mM
gammaATP, 3 mM DTT, 0.03 mM Na-orthovanadate) is added. 0.25 .mu.Ci
33P-gamma-ATP and the kinase described are added in a concentration
of no more than 1 g/ml (a titration should first be performed). In
the presence of the potential inhibitor (e.g. small molecules from
a chemical bank) (e.g. 10 .mu.M), the reaction is incubated at 300
for 1 h. The total reaction volume is 100 .mu.l. The reaction is
stopped by the addition of EDTA (pH 7) up to a final concentration
of 80 mM. The samples are then centrifuged, and 50 .mu.l of the
supernatant is applied to p81 cation exchanger paper (manufactured
by Whatman). The filters are then washed 3 times in 200 ml 180 mM
phosphoric acid (every 5 mins), and once in 200 ml 96% ethanol.
Following drying in air, the radioactivity of the filters is
determined by scintillation counting. Substances that reduce kinase
activity by >=50% (at 10 .mu.M) stand out as a result of a
>50% reduction in incorporation. The specificity and sensitivity
of the possible inhibitors are determined by titration to determine
IC50, and by substitution of other kinases in the assay. Relative
comparisons of the inhibition effect on other kinases thus allow
for a measure of specificity. For the performance of screens on
kinase inhibitors, more modern systems with the scintillation
proximity assay (SPA) also present themselves (manufactured by
Amersham Life Science, MAP kinase SPA; (Zhang et al., Proc Natl
Acad Sci USA, 96, 8511-5, (1999)), (Ichijo et al., Science, 275,
90-4, (1997)). McDonald describes an assay set-up for the
Raf/MEK/ERK cascade that can identify inhibitors of the entire
cascade. A biotinylated peptide which, following phosphorylation
with .sup.33P, can bind to avidin-coated SPA beads is used here.
The MAP cascade is here reconstituted in vitro, expressed with the
individual constituents as GST fusion protein in E. coli or, in the
case of cRAF1, produced with the Baculovirus system. The first
element of the cascade (MAP-KKK) must in this connection always be
uniformly activated to be able to screen inhibitors reliably. This
was achieved in this case by coexpression of src in the Baculovirus
system. A ras-analogous activation of cRaf is thereby achieved.
Another way of activating a MAP kinase by phosphorylation is also
conceivable in principle. Another possibility of constitutive
activation of a MAP kinase consists in mutation of the amino acids
to be phosphorylated. This was, for example, possible in the case
of MEK1 via the replacement of the serines Ser218 and Ser222 by
glutamate (Zhang et al., Proc Natl Acad Sci USA, 96, 8511-5,
(1999)), (Ichijo et al., Science, 275, 90-4, (1997)). In the case
of 9B5, an interaction screen (e.g. y2h system) can firstly be
performed to identify a phosphorylation target or MBP (myelin basic
protein) directly used as substrate. A peptide from the target
sequence can then be synthesised with a biotin anchor. This can
bind to avidin-coupled SPA beads manufactured by Amersham. Purified
(e.g. bacterially produced) 9B5 protein and gamma-32P-ATP are still
added during the reaction. This can take place on a microtitre
scale. Under normal conditions, the target peptide is
phosphorylated and will trigger a scintillation response. From a
library of chemical substances, those with the reaction will now be
pre-incubated prior to addition of the target peptide. Following
addition of the target peptide, the scintillation response can then
be measured. A drop in response signifies the presence of a
potential inhibitor. The library searched should be as complex as
possible, and contain many different substance classes. Examples of
a substance class-specific inhibitor for a protein kinase (p38) can
be found for example in U.S. Pat. No. 5,945,418. Other substance
classes that can inhibit protein kinases are for example bis
monocyclic, bicyclic or heterocyclic aryl compounds (WO 92/20642),
vinylene-azaindol derivatives (WO 94/14808),
1-cyclopropyl-4-pyridyl-quinolone (U.S. Pat. No. 5,330,992), styryl
compounds (U.S. Pat. No. 5,217,999), styryl-substituted pyridyl
compounds (U.S. Pat. No. 5,302,606), certain quinazoline
derivatives (EP Application No. 0 566 266 A1), Selenoindoles and
selenides (WO 94/03427), tricyclic polyhydroxylic compounds (WO
92/21660), and benzylphosphonic acids (WO 91/15495). The specific
nature of the assay is solely determined by the identity of the
Ser/Thr kinase described. Various substrates can be used. These may
be used in various forms of the aforementioned assay principle. The
essential point in this is, however, always the use of the kinase
activity of 9B5 for screening purposes. For example, the substrate
may occur in free solution as well as being coupled to a phase, as
mentioned above. The substrate may, however, also be borne on a
cell surface or even be presented intracellularly. This does not
affect the underlying assay principle.
Example 7
Production of Transgenic and Knock-Out Mice
[0111] Knowledge of the sequence of 9B5 can be used to produce
genetically modified mice (or other animals). To this end, for
example a constitutively inactive mutant of 9B5 is expressed in
transgenic mice, e.g. with an NSE promoter in neurons, with an MBP
promoter in oligodendrocytes, etc. This should have a
dominant-negative effect, and thus imitate inhibition of 9B5. This
may yield valuable indications of possible pharmacological effects
of inhibitors in vivo. A constitutively active kinase can also be
expressed.
[0112] The production of knock-out animals may also yield
indications of the effects of inhibitors. The production of these
genetically modified mice or other animals takes place in
accordance with standard methods familiar to the expert.
[0113] The genetically modified animals can then be investigated in
various disease models (e.g. experimentally induced stroke, MCAO),
or tumour models.
Example 8
Proliferation Assays
[0114] Many of the previously known serine threonine kinases are
involved in neoplastic processes. The involvement of 9B5 in the
growth of cells, the cell cycle and tumorigenic transformation can
be further investigated by transfecting expression constructs with
9B5 in eukaryotic cells and then investigating the induction of
tumorigenicity, e.g. in a Soft-Agar test (Zhang et al., Proc Natl
Acad Sci USA, 96, 8511-5, (1999)), (Ichijo et al., Science, 275,
90-4, (1997)). On cell types, several customary lines, e.g. Cos
cells, HEK cells, PC12 cells, THP-1 cells and primary cells, such
as for example neurons and astrocytes, are considered, as are other
immortalised and primary cell lines, as required.
Sequence CWU 1
1
2113170DNAMus musculus 1aaagggccgt cctggtccag ccgttccctg ggtgcccgtt
gccggaactc tatcgcttcc 60tgccctgagg aacaacccca tgtgggcaac tataggctgc
taaggaccat cgggaagggc 120aacttcgcca aagtcaagct ggctcggcat
atcctcacgg gccgggaggt cgctattaag 180atcattgata agacccagct
gaaccccagt agcttgcaga agctgttcag agaagtccga 240attatgaagg
gactcaacca ccccaacatc gtgaagcttt ttgaggtgat agagacggag
300aagacgctat acctggtgat ggaatacgct agcgcaggag aagtgtttga
ctacctcgtg 360tcgcacggcc gcatgaagga gaaggaggct cgagccaagt
tgcggcagat cgtgtcagcc 420gtgcactact gtcatcagaa gaacattgta
cacagggatc taaaggctga aaacctgttg 480ctggatgccg aggccaacat
caaaatcgcc gacttcggct tcagcaatga gttcacgctg 540ggctccaagc
tggacacctt ctgtgggagc cccccatacg ccgccccaga gctgttccag
600ggcaagaagt atgatgggcc agaggtggac atctggagcc tgggtgtcat
cctgtacacg 660ctggtcagcg gctccctgcc cttcgatggg cacaacctca
aggagctgcg ggagcgaatc 720ctcagaggaa agtaccgggt ccccttctac
atgtctacag actgcgagag cattctgcgg 780agatttctgg tgctgaaccc
cgcaaaacgc tgtactctgg agcaaatcat gaaagacaaa 840tggatcaaca
tcggctatga gggtgaggag ctgaagccat acacggagcc tgaggaggac
900ttcggggaca ccaagagaat tgaggtgatg gtgggtatgg gctacacacg
ggaagaaatc 960aaagaggcct tgaccaacca gaagtacaac gaggtgaccg
ccacctacct cctgctgggc 1020aggaagactg atgagggtgg ggaccggggt
gccccagggc tggccctggc acgggtgcgg 1080gcgcccagcg acaccaccaa
cgggacaagc tccagcaaag gcagcagcca caacaaaggg 1140caacgggctt
cttcctccac ctaccaccgc cagcgccgtc acagtgactt ctgtggcccg
1200tcccctgccc cgctgcaccc gaagcgcacg ccaaccagca cgggagacac
ggagctcaaa 1260gaagagcgga tgccgggtcg gaaagcgagc tgcagtgcag
tgggcagtgg aagtcgaggc 1320ttgcccccct ccagccccat ggtcagcagt
gcccacaacc ccaataaggc agagatccct 1380gagcggcgga aggacagcac
tagcacccct aacaacctcc cccccagcat gatgacccga 1440agaaacacct
atgtgtgcac agagcgacca ggatctgaac gcccgtcctt gttgccaaat
1500ggcaaagaaa atagctccgg tacctcgcgg gtgccccctg cctcgccttc
cagtcatagc 1560ctggctcccc cgtcaggcga gcggagccgc ctggctcggg
gctccaccat ccgcagcacc 1620ttccatgggg gccaggtccg agaccggcgg
gcagggagcg ggagtggcgg gggtgtgcag 1680aatggacccc cagcctcacc
cacgcttgcc cacgaggccg cacccctgcc ctccgggcgg 1740cctcgcccca
ccaccaacct cttcaccaag ctgacctcca aactgacccg aagggtcaca
1800gacgaacctg agagaatcgg gggacctgag gtcacaagtt gccatctacc
ttgggataaa 1860acggaaaccg cccccaggct gctccgattc ccctggagtg
tgaagctgac cagctcgcga 1920cctcctgagg ccctgatggc tgccatgcga
caggccacag cggccgcccg ctgccggtgc 1980cgccagccgc agccgttcct
gctggcctgc ctgcacgggg gtgcgggcgg gcccgagccc 2040ctgtcccatt
tcgaagtgga ggtgtgccag ctgccccggc ccggcctcag gggcgtcctc
2100ttccgccgtg tggcgggcac cgccctggcc ttccgcaccc tcgtcacccg
catctccaac 2160gacctcgaac tctgagccac cgccaccact accaccgcca
cagccaccat cacagcccgg 2220gtcccttctt tctctggttc ctttcacttc
cccaagaggg gaagaggaca gaggagaggg 2280tgccctgtgt catgactgaa
gtttccctgg attagattgg tggacagaga cagtgtgggg 2340acacatgaca
tgataagagg gctcagcagg gggagctggc accctcctag ggcctctggt
2400gggacccccc tccccacaat cttgttcttc tgcagggcac ctgaggagac
tttggggaca 2460ggagtgagaa gggaaactga ggaaattctc ccattcaggg
agagctgcca ggattaatga 2520ctggagacag acttgggggg ttcagggagt
tgggggagtc acagacagaa accttcccct 2580cactccccct tatgatcgaa
cctccttcat gccccaggct ggcgcggggc actttgtaca 2640aatccgtgta
tatactcctg tccctctgca gaggtctctc ggggagctgc tgctgccgcc
2700tccgattttt aagttattgc cccgcccctt ctgtcagctc ctcatctgca
gcctgttact 2760caataaacag taggagtccc tccaaccccg acctcctccc
tggccgacct ggggtttccc 2820ttctcagccc ttggcctgca ggtgagccag
ggagctgggg acttgacccc aacctgtggt 2880tctgcttgct gagcctttgt
tatctcatct tcagaatggg aacagtgggg ttggaggatg 2940ggtcaaggat
gactatggaa gagggcagaa cagagctcag cctcttccac gaggccccag
3000ccttctgtga caccctcctc ttggccactc actcccctct gccatattac
actggaccca 3060gagcctcttc ctattccagt aatacatgta ttcaataaac
aatcaacgac tggtgccgac 3120tccacgctag gcccagttct ggacataaaa
aaaaaaaaaa aaaaaaaaaa 317023250DNAMus musculus 2aaagggccgt
cctggtccag ccgttccctg ggtgcccgtt gccggaactc tatcgcttcc 60tgccctgagg
aacaacccca tgtgggcaac tataggctgc taaggaccat cgggaagggc
120aacttcgcca aagtcaagct ggctcggcat atcctcacgg gccgggaggt
cgctattaag 180atcattgata agacccagct gaaccccagt agcttgcaga
agctgttcag agaagtccga 240attatgaagg gactcaacca ccccaacatc
gtgaagcttt ttgaggtgat agagacggag 300aagacgctat acctggtgat
ggaatacgct agcgcaggag aagtgtttga ctacctcgtg 360tcgcacggcc
gcatgaagga gaaggaggct cgagccaagt tgcggcagat cgtgtcagcc
420gtgcactact gtcatcagaa gaacattgta cacagggatc taaaggctga
aaacctgttg 480ctggatgccg aggccaacat caaaatcgcc gacttcggct
tcagcaatga gttcacgctg 540ggctccaagc tggacacctt ctgtgggagc
cccccatacg ccgccccaga gctgttccag 600ggcaagaagt atgatgggcc
agaggtggac atctggagcc tgggtgtcat cctgtacacg 660ctggtcagcg
gctccctgcc cttcgatggg cacaacctca aggagctgcg ggagcgaatc
720ctcagaggaa agtaccgggt ccccttctac atgtctacag actgcgagag
cattctgcgg 780agatttctgg tgctgaaccc cgcaaaacgc tgtactctgg
agcaaatcat gaaagacaaa 840tggatcaaca tcggctatga gggtgaggag
ctgaagccat acacggagcc tgaggaggac 900ttcggggaca ccaagagaat
tgaggtgatg gtgggtatgg gctacacacg ggaagaaatc 960aaagaggcct
tgaccaacca gaagtacaac gaggtgaccg ccacctacct cctgctgggc
1020aggaagactg atgagggtgg ggaccggggt gccccagggc tggccctggc
acgggtgcgg 1080gcgcccagcg acaccaccaa cgggacaagc tccagcaaag
gcagcagcca caacaaaggg 1140caacgggctt cttcctccac ctaccaccgc
cagcgccgtc acagtgactt ctgtggcccg 1200tcccctgccc cgctgcaccc
gaagcgcagc ccaaccagca cgggagacac ggagctcaaa 1260gaagagcgga
tgccgggtcg gaaagcgagc tgcagtgcag tgggcagtgg aagtcgaggc
1320ttgcccccct ccagccccat ggtcagcagt gcccacaacc ccaataaggc
agagatccct 1380gagcggcgga aggacagcac tagcacccct aacaacctcc
cccccagcat gatgacccga 1440agaaacacct atgtgtgcac agagcgacca
ggatctgaac gcccgtcctt gttgccaaat 1500ggcaaagaaa atagctccgg
tacctcgcgg gtgccccctg cctcgccttc cagtcatagc 1560ctggctcccc
cgtcaggcga gcggagccgc ctggctcggg gctccaccat ccgcagcacc
1620ttccatgggg gccaggtccg agaccggcgg gcagggagcg ggagtggcgg
gggtgtgcag 1680aatggacccc cagcctcacc cacgcttgcc cacgaggccg
cacccctgcc ctccgggcgg 1740cctcgcccca ccaccaacct cttcaccaag
ctgacctcca aactgacccg aagggttacc 1800ctcgatccct ctaaacggca
gaactctaac cgctgtgtct cgggcgcctc tctgccccag 1860ggatccaaaa
tcaggtcaca gacgaacctg agagaatcgg gggacctgag gtcacaagtt
1920gccatctacc ttgggataaa acggaaaccg cccccaggct gctccgattc
ccctggagtg 1980tgaagctgac cagctcgcga cctcctgagg ccctgatggc
tgccatgcga caggccacag 2040cggccgcccg ctgccggtgc cgccagccgc
agccgttcct gctggcctgc ctgcacgggg 2100gtgcgggcgg gcccgagccc
ctgtcccatt tcgaagtgga ggtgtgccag ctgccccggc 2160ccggcctcag
gggcgtcctc ttccgccgtg tggcgggcac cgccctggcc ttccgcaccc
2220tcgtcacccg catctccaac gacctcgaac tctgagccac cgccaccact
accaccgcca 2280cagccaccat cacagcccgg gtcccttctt tctctggttc
ctttcacttc cccaagaggg 2340gaagaggaca gaggagaggg tgccctgtgt
catgactgaa gtttccctgg attagattgg 2400tggacagaga cagtgtgggg
acacatgaca tgataagagg gctcagcagg gggagctggc 2460accctcctag
ggcctctggt gggacccccc tccccacaat cttgttcttc tgcagggcac
2520ctgaggagac tttggggaca ggagtgagaa gggaaactga ggaaattctc
ccattcaggg 2580agagctgcca ggattaatga ctggagacag acttgggggg
ttcagggagt tgggggagtc 2640acagacagaa accttcccct cactccccct
tatgatcgaa cctccttcat gccccaggct 2700ggcgcggggc actttgtaca
aatccgtgta tatactcctg tccctctgca gaggtctctc 2760ggggagctgc
tgctgccgcc tccgattttt aagttattgc cccgcccctt ctgtcagctc
2820ctcatctgca gcctgttact caataaacag taggagtccc tccaaccccg
acctcctccc 2880tggccgacct ggggtttccc ttctcagccc ttggcctgca
ggtgagccag ggagctgggg 2940acttgacccc aacctgtggt tctgcttgct
gagcctttgt tatctcatct tcagaatggg 3000aacagtgggg ttggaggatg
ggtcaaggat gactatggaa gagggcagaa cagagctcag 3060cctcttccac
gaggccccag ccttctgtga caccctcctc ttggccactc actcccctct
3120gccatattac actggaccca gagcctcttc ctattccagt aatacatgta
ttcaataaac 3180aatcaacgac tggtgccgac tccacgctag gcccagttct
ggacataaaa aaaaaaaaaa 3240aaaaaaaaaa 325033312DNAHomo sapiens
3aggggaccct gggacccccg ccccccccac ccggccgccc ctgccccccg ggacccggag
60aagatgtctt cgcggacggt gctggccccg ggcaacgatc ggaactcgga cacgcatggc
120accttgggca gtggccgctc ctcggacaaa ggcccgtcct ggtccagccg
ctcactgggt 180gcccgttgcc ggaactccat cgcctcctgt cccgaggagc
agccccacgt gggcaactac 240cgcctgctga ggaccattgg gaagggcaac
tttgccaaag tcaagctggc tcggcacatc 300ctcactggtc gggaggttgc
catcaagatt atcgacaaaa cccagctgaa tcccagcagc 360ctgcagaagc
tgttccgaga agtccgcatc atgaagggcc taaaccaccc caacatcgtg
420aagctctttg aggtgattga gactgagaag acgctgtacc tggtgatgga
gtacgcaagt 480gctggagaag tgtttgacta cctcgtgtcg catggccgca
tgaaggagaa ggaagctcga 540gccaagttcc gacagattgt ttcggctgtg
cactattgtc accagaaaaa tattgtacac 600agggacctga aggctgagaa
cctcttgctg gatgccgagg ccaacatcaa gattgctgac 660tttggcttca
gcaacgagtt cacgctggga tcgaagctgg acacgttctg cgggagcccc
720ccatatgccg ccccggagct gtttcagggc aagaagtacg acgggccgga
ggtggacatc 780tggagcctgg gagtcatcct gtacaccctc gtcagcggct
ccctgccctt cgacgggcac 840aacctcaagg agctgcggga gcgagtactc
agagggaagt accgggtccc tttctacatg 900tcaacagact gtgagagcat
cctgcggaga tttttggtgc tgaacccagc taaacgctgt 960actctcgagc
aaatcatgaa agacaaatgg atcaacatcg gctatgaggg tgaggagttg
1020aagccataca cagagcccga ggaggacttc ggggacacca agagaattga
ggtgatggtg 1080ggtatgggct acacacggga agaaatcaaa gagtccttga
ccagccagaa gtacaacgaa 1140gtgaccgcca cctacctcct gctgggcagg
aagactgagg agggtgggga ccggggcgcc 1200ccagggctgg ccctggcacg
ggtgcgggcg cccagcgaca ccaccaacgg aacaagttcc 1260agcaaaggca
ccagccacag caaagggcag cggagttcct cttccaccta ccaccgccag
1320cgcaggcata gcgatttctg tggcccatcc cctgcacccc tgcaccccaa
acgcagcccg 1380acgagcacgg gggaggcgga gctgaaggag gagcggctgc
caggccggaa ggcgagctgc 1440agcaccgcgg ggagtgggag tcgagggctg
cccccctcca gccccatggt cagcagcgcc 1500cacaacccca acaaggcaga
gatcccagag cggcggaagg acagcacgag cacccccaac 1560aacctccctc
ctagcatgat gacccgcaga aacacctacg tttgcacaga acgcccgggg
1620gctgagcgcc cgtcactgtt gccaaatggg aaagaaaaca gctcaggcac
cccacgggtg 1680ccccctgcct ccccctccag tcacagcctg gcacccccat
caggggagcg gagccgcctg 1740gcacgtggtt ccaccatccg cagcaccttc
catggtggcc aggtccggga ccggcgggca 1800gggggtgggg gtggtggggg
tgtgcagaat gggccccctg cctctcccac actggcccat 1860gaggctgcac
ccctgcccgc cgggcggccc cgccccacca ccaacctctt caccaagctg
1920acctccaaac tgacccgaag ggtcgcagac gaacctgaga gaatcggggg
acctgaggtc 1980acaagttgcc atctaccttg ggatcaaacg gaaaccgccc
cccggctgct ccgattcccc 2040tggagtgtga agctgaccag ctcgcgccct
cctgaggccc tgatggcagc tctgcgccag 2100gccacagcag ccgcccgctg
ccgctgccgc cagccacagc cgttcctgct ggcctgcctg 2160cacgggggtg
cgggcgggcc cgagcccctg tcccacttcg aagtggaggt ctgccagctg
2220ccccggccag gcttgcgggg agttctcttc cgccgtgtgg cgggcaccgc
cctggccttc 2280cgcaccctcg tcacccgcat ctccaacgac ctcgagctct
gagccaccac ggtcccaggg 2340cccttactct tcctctccct tgtcgccttc
acttctacag gaggggaagg ggccagggag 2400gggattctcc ctttatcatc
acctcagttt ccctgaatta tatttggggg caaagattgt 2460cccctctgct
gttctctggg gccgctcagc acagaagaag gatgaggggg ctcagcgggg
2520ggagctggca ccttcctgga gcctccagcc agtcctgtcc tccctcgccc
taccaagagg 2580gcacctgagg agactttggg gacagggcag gggcagggag
ggaaactgag gaaatcttcc 2640attcctccca acagctcaaa attaggcctt
gggcaggggc agggagagct gctgagccta 2700aagactggag aatctggggg
actgggagtg ggggtcagag aggcagattc cttcccctcc 2760cgtcccctca
cgctcaaacc cccacttcct gccccaggct ggcgcggggc actttgtaca
2820aatccttgta aataccccac accctcccct ctgcaaaggt ctcttgagga
gctgccgctg 2880tcacctacgg tttttaagtt attacacccc gaccctcctc
ctgtcagccc cctcacctgc 2940agcctgttgc ccaataaatt taggagagtc
cccccctccc caatgctgac cctaggattt 3000tccttccctg ccctcacctg
caaatgagtt aaagaagagg cgtgggaatc caggcagtgg 3060tttttccttt
cggagcctcg gttttctcat ctgcagaatg ggagcggtgg gggtgggaag
3120gtaaggatgg tcgtggaaga aggcaggatg gaactcggcc tcatccccga
ggccccagtt 3180cctatatcgg gccccccatt catccactca cactcccagc
caccatgtta cactggactc 3240taagccactt cttactccag tagtaaattt
attcaataaa caatcattga cccatgccta 3300aaaaaaaaaa aa 331243392DNAHomo
sapiens 4aggggaccct gggacccccg ccccccccac ccggccgccc ctgccccccg
ggacccggag 60aagatgtctt cgcggacggt gctggccccg ggcaacgatc ggaactcgga
cacgcatggc 120accttgggca gtggccgctc ctcggacaaa ggcccgtcct
ggtccagccg ctcactgggt 180gcccgttgcc ggaactccat cgcctcctgt
cccgaggagc agccccacgt gggcaactac 240cgcctgctga ggaccattgg
gaagggcaac tttgccaaag tcaagctggc tcggcacatc 300ctcactggtc
gggaggttgc catcaagatt atcgacaaaa cccagctgaa tcccagcagc
360ctgcagaagc tgttccgaga agtccgcatc atgaagggcc taaaccaccc
caacatcgtg 420aagctctttg aggtgattga gactgagaag acgctgtacc
tggtgatgga gtacgcaagt 480gctggagaag tgtttgacta cctcgtgtcg
catggccgca tgaaggagaa ggaagctcga 540gccaagttcc gacagattgt
ttcggctgtg cactattgtc accagaaaaa tattgtacac 600agggacctga
aggctgagaa cctcttgctg gatgccgagg ccaacatcaa gattgctgac
660tttggcttca gcaacgagtt cacgctggga tcgaagctgg acacgttctg
cgggagcccc 720ccatatgccg ccccggagct gtttcagggc aagaagtacg
acgggccgga ggtggacatc 780tggagcctgg gagtcatcct gtacaccctc
gtcagcggct ccctgccctt cgacgggcac 840aacctcaagg agctgcggga
gcgagtactc agagggaagt accgggtccc tttctacatg 900tcaacagact
gtgagagcat cctgcggaga tttttggtgc tgaacccagc taaacgctgt
960actctcgagc aaatcatgaa agacaaatgg atcaacatcg gctatgaggg
tgaggagttg 1020aagccataca cagagcccga ggaggacttc ggggacacca
agagaattga ggtgatggtg 1080ggtatgggct acacacggga agaaatcaaa
gagtccttga ccagccagaa gtacaacgaa 1140gtgaccgcca cctacctcct
gctgggcagg aagactgagg agggtgggga ccggggcgcc 1200ccagggctgg
ccctggcacg ggtgcgggcg cccagcgaca ccaccaacgg aacaagttcc
1260agcaaaggca ccagccacag caaagggcag cggagttcct cttccaccta
ccaccgccag 1320cgcaggcata gcgatttctg tggcccatcc cctgcacccc
tgcaccccaa acgcagcccg 1380acgagcacgg gggaggcgga gctgaaggag
gagcggctgc caggccggaa ggcgagctgc 1440agcaccgcgg ggagtgggag
tcgagggctg cccccctcca gccccatggt cagcagcgcc 1500cacaacccca
acaaggcaga gatcccagag cggcggaagg acagcacgag cacccccaac
1560aacctccctc ctagcatgat gacccgcaga aacacctacg tttgcacaga
acgcccgggg 1620gctgagcgcc cgtcactgtt gccaaatggg aaagaaaaca
gctcaggcac cccacgggtg 1680ccccctgcct ccccctccag tcacagcctg
gcacccccat caggggagcg gagccgcctg 1740gcacgtggtt ccaccatccg
cagcaccttc catggtggcc aggtccggga ccggcgggca 1800gggggtgggg
gtggtggggg tgtgcagaat gggccccctg cctctcccac actggcccat
1860gaggctgcac ccctgcccgc cgggcggccc cgccccacca ccaacctctt
caccaagctg 1920acctccaaac tgacccgaag ggttaccctc gatccctcta
aacggcagaa ctctaaccgc 1980tgtgtttcgg gcgcctctct gccccaggga
tccaagatca ggtcgcagac gaacctgaga 2040gaatcggggg acctgaggtc
acaagttgcc atctaccttg ggatcaaacg gaaaccgccc 2100cccggctgct
ccgattcccc tggagtgtga agctgaccag ctcgcgccct cctgaggccc
2160tgatggcagc tctgcgccag gccacagcag ccgcccgctg ccgctgccgc
cagccacagc 2220cgttcctgct ggcctgcctg cacgggggtg cgggcgggcc
cgagcccctg tcccacttcg 2280aagtggaggt ctgccagctg ccccggccag
gcttgcgggg agttctcttc cgccgtgtgg 2340cgggcaccgc cctggccttc
cgcaccctcg tcacccgcat ctccaacgac ctcgagctct 2400gagccaccac
ggtcccaggg cccttactct tcctctccct tgtcgccttc acttctacag
2460gaggggaagg ggccagggag gggattctcc ctttatcatc acctcagttt
ccctgaatta 2520tatttggggg caaagattgt cccctctgct gttctctggg
gccgctcagc acagaagaag 2580gatgaggggg ctcagcgggg ggagctggca
ccttcctgga gcctccagcc agtcctgtcc 2640tccctcgccc taccaagagg
gcacctgagg agactttggg gacagggcag gggcagggag 2700ggaaactgag
gaaatcttcc attcctccca acagctcaaa attaggcctt gggcaggggc
2760agggagagct gctgagccta aagactggag aatctggggg actgggagtg
ggggtcagag 2820aggcagattc cttcccctcc cgtcccctca cgctcaaacc
cccacttcct gccccaggct 2880ggcgcggggc actttgtaca aatccttgta
aataccccac accctcccct ctgcaaaggt 2940ctcttgagga gctgccgctg
tcacctacgg tttttaagtt attacacccc gaccctcctc 3000ctgtcagccc
cctcacctgc agcctgttgc ccaataaatt taggagagtc cccccctccc
3060caatgctgac cctaggattt tccttccctg ccctcacctg caaatgagtt
aaagaagagg 3120cgtgggaatc caggcagtgg tttttccttt cggagcctcg
gttttctcat ctgcagaatg 3180ggagcggtgg gggtgggaag gtaaggatgg
tcgtggaaga aggcaggatg gaactcggcc 3240tcatccccga ggccccagtt
cctatatcgg gccccccatt catccactca cactcccagc 3300caccatgtta
cactggactc taagccactt cttactccag tagtaaattt attcaataaa
3360caatcattga cccatgccta aaaaaaaaaa aa 33925724PRTMus musculus
5Lys Gly Pro Ser Trp Ser Ser Arg Ser Leu Gly Ala Arg Cys Arg Asn1 5
10 15Ser Ile Ala Ser Cys Pro Glu Glu Gln Pro His Val Gly Asn Tyr
Arg20 25 30Leu Leu Arg Thr Ile Gly Lys Gly Asn Phe Ala Lys Val Lys
Leu Ala35 40 45Arg His Ile Leu Thr Gly Arg Glu Val Ala Ile Lys Ile
Ile Asp Lys50 55 60Thr Gln Leu Asn Pro Ser Ser Leu Gln Lys Leu Phe
Arg Glu Val Arg65 70 75 80Ile Met Lys Gly Leu Asn His Pro Asn Ile
Val Lys Leu Phe Glu Val85 90 95Ile Glu Thr Glu Lys Thr Leu Tyr Leu
Val Met Glu Tyr Ala Ser Ala100 105 110Gly Glu Val Phe Asp Tyr Leu
Val Ser His Gly Arg Met Lys Glu Lys115 120 125Glu Ala Arg Ala Lys
Leu Arg Gln Ile Val Ser Ala Val His Tyr Cys130 135 140His Gln Lys
Asn Ile Val His Arg Asp Leu Lys Ala Glu Asn Leu Leu145 150 155
160Leu Asp Ala Glu Ala Asn Ile Lys Ile Ala Asp Phe Gly Phe Ser
Asn165 170 175Glu Phe Thr Leu Gly Ser Lys Leu Asp Thr Phe Cys Gly
Ser Pro Pro180 185 190Tyr Ala Ala Pro Glu Leu Phe Gln Gly Lys Lys
Tyr Asp Gly Pro Glu195 200 205Val Asp Ile Trp Ser Leu Gly Val Ile
Leu Tyr Thr Leu Val Ser Gly210 215 220Ser Leu Pro Phe Asp Gly His
Asn Leu Lys Glu Leu Arg Glu Arg Ile225 230 235 240Leu Arg Gly Lys
Tyr Arg Val Pro Phe Tyr Met Ser Thr Asp Cys Glu245 250 255Ser Ile
Leu Arg Arg Phe Leu Val Leu Asn Pro Ala Lys Arg Cys Thr260 265
270Leu Glu Gln Ile Met Lys Asp Lys Trp Ile Asn Ile Gly Tyr Glu
Gly275 280 285Glu Glu Leu Lys Pro Tyr Thr Glu Pro Glu Glu Asp Phe
Gly Asp Thr290
295 300Lys Arg Ile Glu Val Met Val Gly Met Gly Tyr Thr Arg Glu Glu
Ile305 310 315 320Lys Glu Ala Leu Thr Asn Gln Lys Tyr Asn Glu Val
Thr Ala Thr Tyr325 330 335Leu Leu Leu Gly Arg Lys Thr Asp Glu Gly
Gly Asp Arg Gly Ala Pro340 345 350Gly Leu Ala Leu Ala Arg Val Arg
Ala Pro Ser Asp Thr Thr Asn Gly355 360 365Thr Ser Ser Ser Lys Gly
Ser Ser His Asn Lys Gly Gln Arg Ala Ser370 375 380Ser Ser Thr Tyr
His Arg Gln Arg Arg His Ser Asp Phe Cys Gly Pro385 390 395 400Ser
Pro Ala Pro Leu His Pro Lys Arg Thr Pro Thr Ser Thr Gly Asp405 410
415Thr Glu Leu Lys Glu Glu Arg Met Pro Gly Arg Lys Ala Ser Cys
Ser420 425 430Ala Val Gly Ser Gly Ser Arg Gly Leu Pro Pro Ser Ser
Pro Met Val435 440 445Ser Ser Ala His Asn Pro Asn Lys Ala Glu Ile
Pro Glu Arg Arg Lys450 455 460Asp Ser Thr Ser Thr Pro Asn Asn Leu
Pro Pro Ser Met Met Thr Arg465 470 475 480Arg Asn Thr Tyr Val Cys
Thr Glu Arg Pro Gly Ser Glu Arg Pro Ser485 490 495Leu Leu Pro Asn
Gly Lys Glu Asn Ser Ser Gly Thr Ser Arg Val Pro500 505 510Pro Ala
Ser Pro Ser Ser His Ser Leu Ala Pro Pro Ser Gly Glu Arg515 520
525Ser Arg Leu Ala Arg Gly Ser Thr Ile Arg Ser Thr Phe His Gly
Gly530 535 540Gln Val Arg Asp Arg Arg Ala Gly Ser Gly Ser Gly Gly
Gly Val Gln545 550 555 560Asn Gly Pro Pro Ala Ser Pro Thr Leu Ala
His Glu Ala Ala Pro Leu565 570 575Pro Ser Gly Arg Pro Arg Pro Thr
Thr Asn Leu Phe Thr Lys Leu Thr580 585 590Ser Lys Leu Thr Arg Arg
Val Thr Asp Glu Pro Glu Arg Ile Gly Gly595 600 605Pro Glu Val Thr
Ser Cys His Leu Pro Trp Asp Lys Thr Glu Thr Ala610 615 620Pro Arg
Leu Leu Arg Phe Pro Trp Ser Val Lys Leu Thr Ser Ser Arg625 630 635
640Pro Pro Glu Ala Leu Met Ala Ala Met Arg Gln Ala Thr Ala Ala
Ala645 650 655Arg Cys Arg Cys Arg Gln Pro Gln Pro Phe Leu Leu Ala
Cys Leu His660 665 670Gly Gly Ala Gly Gly Pro Glu Pro Leu Ser His
Phe Glu Val Glu Val675 680 685Cys Gln Leu Pro Arg Pro Gly Leu Arg
Gly Val Leu Phe Arg Arg Val690 695 700Ala Gly Thr Ala Leu Ala Phe
Arg Thr Leu Val Thr Arg Ile Ser Asn705 710 715 720Asp Leu Glu
Leu6660PRTMus musculus 6Lys Gly Pro Ser Trp Ser Ser Arg Ser Leu Gly
Ala Arg Cys Arg Asn1 5 10 15Ser Ile Ala Ser Cys Pro Glu Glu Gln Pro
His Val Gly Asn Tyr Arg20 25 30Leu Leu Arg Thr Ile Gly Lys Gly Asn
Phe Ala Lys Val Lys Leu Ala35 40 45Arg His Ile Leu Thr Gly Arg Glu
Val Ala Ile Lys Ile Ile Asp Lys50 55 60Thr Gln Leu Asn Pro Ser Ser
Leu Gln Lys Leu Phe Arg Glu Val Arg65 70 75 80Ile Met Lys Gly Leu
Asn His Pro Asn Ile Val Lys Leu Phe Glu Val85 90 95Ile Glu Thr Glu
Lys Thr Leu Tyr Leu Val Met Glu Tyr Ala Ser Ala100 105 110Gly Glu
Val Phe Asp Tyr Leu Val Ser His Gly Arg Met Lys Glu Lys115 120
125Glu Ala Arg Ala Lys Leu Arg Gln Ile Val Ser Ala Val His Tyr
Cys130 135 140His Gln Lys Asn Ile Val His Arg Asp Leu Lys Ala Glu
Asn Leu Leu145 150 155 160Leu Asp Ala Glu Ala Asn Ile Lys Ile Ala
Asp Phe Gly Phe Ser Asn165 170 175Glu Phe Thr Leu Gly Ser Lys Leu
Asp Thr Phe Cys Gly Ser Pro Pro180 185 190Tyr Ala Ala Pro Glu Leu
Phe Gln Gly Lys Lys Tyr Asp Gly Pro Glu195 200 205Val Asp Ile Trp
Ser Leu Gly Val Ile Leu Tyr Thr Leu Val Ser Gly210 215 220Ser Leu
Pro Phe Asp Gly His Asn Leu Lys Glu Leu Arg Glu Arg Ile225 230 235
240Leu Arg Gly Lys Tyr Arg Val Pro Phe Tyr Met Ser Thr Asp Cys
Glu245 250 255Ser Ile Leu Arg Arg Phe Leu Val Leu Asn Pro Ala Lys
Arg Cys Thr260 265 270Leu Glu Gln Ile Met Lys Asp Lys Trp Ile Asn
Ile Gly Tyr Glu Gly275 280 285Glu Glu Leu Lys Pro Tyr Thr Glu Pro
Glu Glu Asp Phe Gly Asp Thr290 295 300Lys Arg Ile Glu Val Met Val
Gly Met Gly Tyr Thr Arg Glu Glu Ile305 310 315 320Lys Glu Ala Leu
Thr Asn Gln Lys Tyr Asn Glu Val Thr Ala Thr Tyr325 330 335Leu Leu
Leu Gly Arg Lys Thr Asp Glu Gly Gly Asp Arg Gly Ala Pro340 345
350Gly Leu Ala Leu Ala Arg Val Arg Ala Pro Ser Asp Thr Thr Asn
Gly355 360 365Thr Ser Ser Ser Lys Gly Ser Ser His Asn Lys Gly Gln
Arg Ala Ser370 375 380Ser Ser Thr Tyr His Arg Gln Arg Arg His Ser
Asp Phe Cys Gly Pro385 390 395 400Ser Pro Ala Pro Leu His Pro Lys
Arg Ser Pro Thr Ser Thr Gly Asp405 410 415Thr Glu Leu Lys Glu Glu
Arg Met Pro Gly Arg Lys Ala Ser Cys Ser420 425 430Ala Val Gly Ser
Gly Ser Arg Gly Leu Pro Pro Ser Ser Pro Met Val435 440 445Ser Ser
Ala His Asn Pro Asn Lys Ala Glu Ile Pro Glu Arg Arg Lys450 455
460Asp Ser Thr Ser Thr Pro Asn Asn Leu Pro Pro Ser Met Met Thr
Arg465 470 475 480Arg Asn Thr Tyr Val Cys Thr Glu Arg Pro Gly Ser
Glu Arg Pro Ser485 490 495Leu Leu Pro Asn Gly Lys Glu Asn Ser Ser
Gly Thr Ser Arg Val Pro500 505 510Pro Ala Ser Pro Ser Ser His Ser
Leu Ala Pro Pro Ser Gly Glu Arg515 520 525Ser Arg Leu Ala Arg Gly
Ser Thr Ile Arg Ser Thr Phe His Gly Gly530 535 540Gln Val Arg Asp
Arg Arg Ala Gly Ser Gly Ser Gly Gly Gly Val Gln545 550 555 560Asn
Gly Pro Pro Ala Ser Pro Thr Leu Ala His Glu Ala Ala Pro Leu565 570
575Pro Ser Gly Arg Pro Arg Pro Thr Thr Asn Leu Phe Thr Lys Leu
Thr580 585 590Ser Lys Leu Thr Arg Arg Val Thr Leu Asp Pro Ser Lys
Arg Gln Asn595 600 605Ser Asn Arg Cys Val Ser Gly Ala Ser Leu Pro
Gln Gly Ser Lys Ile610 615 620Arg Ser Gln Thr Asn Leu Arg Glu Ser
Gly Asp Leu Arg Ser Gln Val625 630 635 640Ala Ile Tyr Leu Gly Ile
Lys Arg Lys Pro Pro Pro Gly Cys Ser Asp645 650 655Ser Pro Gly
Val6607752PRTHomo sapiens 7Met Ser Ser Arg Thr Val Leu Ala Pro Gly
Asn Asp Arg Asn Ser Asp1 5 10 15Thr His Gly Thr Leu Gly Ser Gly Arg
Ser Ser Asp Lys Gly Pro Ser20 25 30Trp Ser Ser Arg Ser Leu Gly Ala
Arg Cys Arg Asn Ser Ile Ala Ser35 40 45Cys Pro Glu Glu Gln Pro His
Val Gly Asn Tyr Arg Leu Leu Arg Thr50 55 60Ile Gly Lys Gly Asn Phe
Ala Lys Val Lys Leu Ala Arg His Ile Leu65 70 75 80Thr Gly Arg Glu
Val Ala Ile Lys Ile Ile Asp Lys Thr Gln Leu Asn85 90 95Pro Ser Ser
Leu Gln Lys Leu Phe Arg Glu Val Arg Ile Met Lys Gly100 105 110Leu
Asn His Pro Asn Ile Val Lys Leu Phe Glu Val Ile Glu Thr Glu115 120
125Lys Thr Leu Tyr Leu Val Met Glu Tyr Ala Ser Ala Gly Glu Val
Phe130 135 140Asp Tyr Leu Val Ser His Gly Arg Met Lys Glu Lys Glu
Ala Arg Ala145 150 155 160Lys Phe Arg Gln Ile Val Ser Ala Val His
Tyr Cys His Gln Lys Asn165 170 175Ile Val His Arg Asp Leu Lys Ala
Glu Asn Leu Leu Leu Asp Ala Glu180 185 190Ala Asn Ile Lys Ile Ala
Asp Phe Gly Phe Ser Asn Glu Phe Thr Leu195 200 205Gly Ser Lys Leu
Asp Thr Phe Cys Gly Ser Pro Pro Tyr Ala Ala Pro210 215 220Glu Leu
Phe Gln Gly Lys Lys Tyr Asp Gly Pro Glu Val Asp Ile Trp225 230 235
240Ser Leu Gly Val Ile Leu Tyr Thr Leu Val Ser Gly Ser Leu Pro
Phe245 250 255Asp Gly His Asn Leu Lys Glu Leu Arg Glu Arg Val Leu
Arg Gly Lys260 265 270Tyr Arg Val Pro Phe Tyr Met Ser Thr Asp Cys
Glu Ser Ile Leu Arg275 280 285Arg Phe Leu Val Leu Asn Pro Ala Lys
Arg Cys Thr Leu Glu Gln Ile290 295 300Met Lys Asp Lys Trp Ile Asn
Ile Gly Tyr Glu Gly Glu Glu Leu Lys305 310 315 320Pro Tyr Thr Glu
Pro Glu Glu Asp Phe Gly Asp Thr Lys Arg Ile Glu325 330 335Val Met
Val Gly Met Gly Tyr Thr Arg Glu Glu Ile Lys Glu Ser Leu340 345
350Thr Ser Gln Lys Tyr Asn Glu Val Thr Ala Thr Tyr Leu Leu Leu
Gly355 360 365Arg Lys Thr Glu Glu Gly Gly Asp Arg Gly Ala Pro Gly
Leu Ala Leu370 375 380Ala Arg Val Arg Ala Pro Ser Asp Thr Thr Asn
Gly Thr Ser Ser Ser385 390 395 400Lys Gly Thr Ser His Ser Lys Gly
Gln Arg Ser Ser Ser Ser Thr Tyr405 410 415His Arg Gln Arg Arg His
Ser Asp Phe Cys Gly Pro Ser Pro Ala Pro420 425 430Leu His Pro Lys
Arg Ser Pro Thr Ser Thr Gly Glu Ala Glu Leu Lys435 440 445Glu Glu
Arg Leu Pro Gly Arg Lys Ala Ser Cys Ser Thr Ala Gly Ser450 455
460Gly Ser Arg Gly Leu Pro Pro Ser Ser Pro Met Val Ser Ser Ala
His465 470 475 480Asn Pro Asn Lys Ala Glu Ile Pro Glu Arg Arg Lys
Asp Ser Thr Ser485 490 495Thr Pro Asn Asn Leu Pro Pro Ser Met Met
Thr Arg Arg Asn Thr Tyr500 505 510Val Cys Thr Glu Arg Pro Gly Ala
Glu Arg Pro Ser Leu Leu Pro Asn515 520 525Gly Lys Glu Asn Ser Ser
Gly Thr Pro Arg Val Pro Pro Ala Ser Pro530 535 540Ser Ser His Ser
Leu Ala Pro Pro Ser Gly Glu Arg Ser Arg Leu Ala545 550 555 560Arg
Gly Ser Thr Ile Arg Ser Thr Phe His Gly Gly Gln Val Arg Asp565 570
575Arg Arg Ala Gly Gly Gly Gly Gly Gly Gly Val Gln Asn Gly Pro
Pro580 585 590Ala Ser Pro Thr Leu Ala His Glu Ala Ala Pro Leu Pro
Ala Gly Arg595 600 605Pro Arg Pro Thr Thr Asn Leu Phe Thr Lys Leu
Thr Ser Lys Leu Thr610 615 620Arg Arg Val Ala Asp Glu Pro Glu Arg
Ile Gly Gly Pro Glu Val Thr625 630 635 640Ser Cys His Leu Pro Trp
Asp Gln Thr Glu Thr Ala Pro Arg Leu Leu645 650 655Arg Phe Pro Trp
Ser Val Lys Leu Thr Ser Ser Arg Pro Pro Glu Ala660 665 670Leu Met
Ala Ala Leu Arg Gln Ala Thr Ala Ala Ala Arg Cys Arg Cys675 680
685Arg Gln Pro Gln Pro Phe Leu Leu Ala Cys Leu His Gly Gly Ala
Gly690 695 700Gly Pro Glu Pro Leu Ser His Phe Glu Val Glu Val Cys
Gln Leu Pro705 710 715 720Arg Pro Gly Leu Arg Gly Val Leu Phe Arg
Arg Val Ala Gly Thr Ala725 730 735Leu Ala Phe Arg Thr Leu Val Thr
Arg Ile Ser Asn Asp Leu Glu Leu740 745 7508688PRTHomo sapiens 8Met
Ser Ser Arg Thr Val Leu Ala Pro Gly Asn Asp Arg Asn Ser Asp1 5 10
15Thr His Gly Thr Leu Gly Ser Gly Arg Ser Ser Asp Lys Gly Pro Ser20
25 30Trp Ser Ser Arg Ser Leu Gly Ala Arg Cys Arg Asn Ser Ile Ala
Ser35 40 45Cys Pro Glu Glu Gln Pro His Val Gly Asn Tyr Arg Leu Leu
Arg Thr50 55 60Ile Gly Lys Gly Asn Phe Ala Lys Val Lys Leu Ala Arg
His Ile Leu65 70 75 80Thr Gly Arg Glu Val Ala Ile Lys Ile Ile Asp
Lys Thr Gln Leu Asn85 90 95Pro Ser Ser Leu Gln Lys Leu Phe Arg Glu
Val Arg Ile Met Lys Gly100 105 110Leu Asn His Pro Asn Ile Val Lys
Leu Phe Glu Val Ile Glu Thr Glu115 120 125Lys Thr Leu Tyr Leu Val
Met Glu Tyr Ala Ser Ala Gly Glu Val Phe130 135 140Asp Tyr Leu Val
Ser His Gly Arg Met Lys Glu Lys Glu Ala Arg Ala145 150 155 160Lys
Phe Arg Gln Ile Val Ser Ala Val His Tyr Cys His Gln Lys Asn165 170
175Ile Val His Arg Asp Leu Lys Ala Glu Asn Leu Leu Leu Asp Ala
Glu180 185 190Ala Asn Ile Lys Ile Ala Asp Phe Gly Phe Ser Asn Glu
Phe Thr Leu195 200 205Gly Ser Lys Leu Asp Thr Phe Cys Gly Ser Pro
Pro Tyr Ala Ala Pro210 215 220Glu Leu Phe Gln Gly Lys Lys Tyr Asp
Gly Pro Glu Val Asp Ile Trp225 230 235 240Ser Leu Gly Val Ile Leu
Tyr Thr Leu Val Ser Gly Ser Leu Pro Phe245 250 255Asp Gly His Asn
Leu Lys Glu Leu Arg Glu Arg Val Leu Arg Gly Lys260 265 270Tyr Arg
Val Pro Phe Tyr Met Ser Thr Asp Cys Glu Ser Ile Leu Arg275 280
285Arg Phe Leu Val Leu Asn Pro Ala Lys Arg Cys Thr Leu Glu Gln
Ile290 295 300Met Lys Asp Lys Trp Ile Asn Ile Gly Tyr Glu Gly Glu
Glu Leu Lys305 310 315 320Pro Tyr Thr Glu Pro Glu Glu Asp Phe Gly
Asp Thr Lys Arg Ile Glu325 330 335Val Met Val Gly Met Gly Tyr Thr
Arg Glu Glu Ile Lys Glu Ser Leu340 345 350Thr Ser Gln Lys Tyr Asn
Glu Val Thr Ala Thr Tyr Leu Leu Leu Gly355 360 365Arg Lys Thr Glu
Glu Gly Gly Asp Arg Gly Ala Pro Gly Leu Ala Leu370 375 380Ala Arg
Val Arg Ala Pro Ser Asp Thr Thr Asn Gly Thr Ser Ser Ser385 390 395
400Lys Gly Thr Ser His Ser Lys Gly Gln Arg Ser Ser Ser Ser Thr
Tyr405 410 415His Arg Gln Arg Arg His Ser Asp Phe Cys Gly Pro Ser
Pro Ala Pro420 425 430Leu His Pro Lys Arg Ser Pro Thr Ser Thr Gly
Glu Ala Glu Leu Lys435 440 445Glu Glu Arg Leu Pro Gly Arg Lys Ala
Ser Cys Ser Thr Ala Gly Ser450 455 460Gly Ser Arg Gly Leu Pro Pro
Ser Ser Pro Met Val Ser Ser Ala His465 470 475 480Asn Pro Asn Lys
Ala Glu Ile Pro Glu Arg Arg Lys Asp Ser Thr Ser485 490 495Thr Pro
Asn Asn Leu Pro Pro Ser Met Met Thr Arg Arg Asn Thr Tyr500 505
510Val Cys Thr Glu Arg Pro Gly Ala Glu Arg Pro Ser Leu Leu Pro
Asn515 520 525Gly Lys Glu Asn Ser Ser Gly Thr Pro Arg Val Pro Pro
Ala Ser Pro530 535 540Ser Ser His Ser Leu Ala Pro Pro Ser Gly Glu
Arg Ser Arg Leu Ala545 550 555 560Arg Gly Ser Thr Ile Arg Ser Thr
Phe His Gly Gly Gln Val Arg Asp565 570 575Arg Arg Ala Gly Gly Gly
Gly Gly Gly Gly Val Gln Asn Gly Pro Pro580 585 590Ala Ser Pro Thr
Leu Ala His Glu Ala Ala Pro Leu Pro Ala Gly Arg595 600 605Pro Arg
Pro Thr Thr Asn Leu Phe Thr Lys Leu Thr Ser Lys Leu Thr610 615
620Arg Arg Val Thr Leu Asp Pro Ser Lys Arg Gln Asn Ser Asn Arg
Cys625 630 635 640Val Ser Gly Ala Ser Leu Pro Gln Gly Ser Lys Ile
Arg Ser Gln Thr645 650 655Asn Leu Arg Glu Ser Gly Asp Leu Arg Ser
Gln Val Ala Ile Tyr Leu660 665 670Gly Ile Lys Arg Lys Pro Pro Pro
Gly Cys Ser Asp Ser Pro Gly Val675 680 68594PRTMus musculus 9Glu
Leu Lys Leu1104PRTartificial sequenceoligopeptide 10Lys Xaa Gly
Ser1114PRTartificial sequenceoligopeptide 11Lys Val Gly
Ser1128PRTMus musculus 12Gly Lys Gly Asn Phe Ala Lys Val1
5138PRTartificial sequenceoligopeptide 13Gly Xaa Gly Xaa Xaa Gly
Xaa Val1 5148PRTartificial sequenceoligopeptide 14His Arg Asp Leu
Lys Xaa Xaa Asn1 5158PRTMus musculus 15His Arg Asp Leu Lys Ala Glu
Asn1 51618DNAartificial sequenceoligonucleotide 16gttgccatca
agattatc 181720DNAartificial sequenceoligonucleotide 17catgatttgc
tcgagagtac 201820DNAartificial sequenceoligonucleotide 18accccaccgt
gttcttcgac
201920DNAartificial sequenceoligonucleotide 19catttgccat ggacaagatg
202023DNAartificial sequenceoligonucleotide 20tatgatcgaa cctccttcat
gcc 232122DNAartificial sequenceoligonucleotide 21atgtccagaa
ctgggcctag cg 22
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References