U.S. patent application number 10/494010 was filed with the patent office on 2005-04-14 for mnk kinase homologous proteins involved in the regulation of energy homeostasis and organelle metabolism.
Invention is credited to Belgore, Funmi, Bronner, Gunter, Ciossek, Thomas, Eulenberg, Karsten, Jakel, Stefan, Meyer, Christoph, Rudolph, Bettina, Rudolph, Dorothea, Steuernagel, Arnd.
Application Number | 20050080026 10/494010 |
Document ID | / |
Family ID | 26076753 |
Filed Date | 2005-04-14 |
United States Patent
Application |
20050080026 |
Kind Code |
A1 |
Steuernagel, Arnd ; et
al. |
April 14, 2005 |
Mnk kinase homologous proteins involved in the regulation of energy
homeostasis and organelle metabolism
Abstract
The present invention discloses Mnk homologous proteins
regulating the energy homeostasis, the metabolism of triglycerides,
and/or is contributing to membrane stability and/or function of
organelles, and polynucleotides, which identify and encode the
proteins disclosed in this invention. The invention also relates to
the use of these sequences in the diagnosis, study, prevention, and
treatment of diseases and disorders related to body-weight
regulation and thermogenesis, for example, but not limited to,
metabolic diseases such as obesity, as well as related disorders
such as eating disorder, cachexia, diabetes mellitus, hypertension,
coronary heart disease, hypercholesterolemia, dyslipidemia,
osteoarthritis, gallstones, and sleep apnea, and disorders related
to ROS defence, such as diabetes mellitus, neurodegenerative
disorders, and cancer, e.g. cancers of the reproductive organs, and
others.
Inventors: |
Steuernagel, Arnd;
(Gottingen, DE) ; Eulenberg, Karsten; (Borenden,
DE) ; Bronner, Gunter; (Gottingen, DE) ;
Ciossek, Thomas; (Ravensburg, DE) ; Rudolph,
Bettina; (Hannover, DE) ; Rudolph, Dorothea;
(Wien, DE) ; Belgore, Funmi; (London, GB) ;
Jakel, Stefan; (Gottingen, DE) ; Meyer,
Christoph; (Gottingen, DE) |
Correspondence
Address: |
ROTHWELL, FIGG, ERNST & MANBECK, P.C.
1425 K STREET, N.W.
SUITE 800
WASHINGTON
DC
20005
US
|
Family ID: |
26076753 |
Appl. No.: |
10/494010 |
Filed: |
August 12, 2004 |
PCT Filed: |
October 29, 2002 |
PCT NO: |
PCT/EP02/12075 |
Current U.S.
Class: |
514/44A ;
435/6.16 |
Current CPC
Class: |
A61P 39/06 20180101;
A61P 7/00 20180101; A61P 1/16 20180101; C12N 9/1205 20130101; A61P
3/06 20180101; A61P 19/02 20180101; Y10S 514/909 20130101; A61P
3/10 20180101; A61P 9/12 20180101; A61K 38/45 20130101; A61P 11/00
20180101; A61P 3/04 20180101; A61P 1/18 20180101; A61P 9/10
20180101; A61P 25/18 20180101; A61P 35/00 20180101; A61P 25/28
20180101; A61P 3/00 20180101 |
Class at
Publication: |
514/044 ;
435/006 |
International
Class: |
A61K 048/00; C12Q
001/68 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 29, 2001 |
EP |
011258126 |
May 17, 2002 |
EP |
020110730 |
Claims
1. A pharmaceutical composition comprising a nucleic acid molecule
of the MAP kinase interacting kinase (Mnk) gene family or a
polypeptide encoded thereby or a fragment or a variant of said
nucleic acid molecule or said polypeptide or an antibody, an
aptamer or another receptor recognizing a nucleic acid molecule of
the Mnk gene family or a polypeptide encoded thereby together with
pharmaceutically acceptable carriers, diluents and/or
adjuvants.
2. The composition of claim 1, wherein the nucleic acid molecule is
a vertebrate or insect Mnk nucleic acid, particularly a human Mnk
homologous nucleic acid, particularly a nucleic acid encoding a Mnk
homologous nucleic acid encloding a Mnk homologous gene on human
chromosome 19 (Mnk2)(Genbank Accession No. XM.sub.--030637,
identical to Genbank Accession No. AF237775.1, and or/Genbank
Accession Number NM.sub.--017572.1) or a human Mnk protein on
chromosome 1 (Mnk1) (Genbank Accession No. XM.sub.--001600,
identical to Genbank Accession No. AB00409.1, and/or Genbank
Accession Number NM, 003684.2) or a fragment thereof or a variant
thereof.
3. The composition of claim 1 or2, wherein said nucleic acid
molecule (a) hybridizes at 50.degree. C. in a solution containing
1.times.SSG and 0.1% SDS to a Mnk nucleic acid or the complementary
strand thereof, particularly a nucleic acid molecule encoding the
amino acid sequences shown in FIG. 3; (b) it is degenerate with
respect to the nucleic acid molecule of (a) (c) encodes a
polypeptide which is at least 85%, preferably at least 90%, more
preferably at least 95%, more preferably at least 98% and up to
99,6% identical to polypeptides shown in FIG. 3; (d) differs from
the nucleic acid molecule of (a) to (c) by mutation and wherein
said mutation causes an alteration, deletion, duplication or
premature stop in the encoded polypeptide.
4. The composition of claim 1, wherein the nucleic acid molecule is
a DNA molecule, particularly a cDNA or a genomic DNA.
5. The composition of claim 1, wherein said nucleic acid encodes a
polypeptide contributing to regulating the energy homeostasis
and/or the metabolism of triglycerides and/or to membrane stability
and/or function in organelles such as mitochondria.
6. The composition of claim 1, wherein said nucleic acid molecule
is a recombinant nucleic acid molecule.
7. The composition of claim 1, wherein the nucleic acid molecule is
a vector, particularly an expression vector.
8. The composition of claim 1, wherein the polypeptide is a
recombinant polypeptide.
9. The composition of claim 8, wherein said recombinant polypeptide
is a fusion polypeptide.
10. The composition of claim 1, wherein said nucleic acid molecule
is selected from hybridization probes, primers and anti-sense
oligonucleotides.
11. The composition of claim 1 which is a diagnostic
composition.
12. The composition of claim 1 which is a pharmaceutical
composition.
13. The composition of claim 1 for the manufacture of an agent for
detecting and/or verifying, for the treatment, alleviation and/or
prevention of diseases and disorders related to body-weight
regulation and thermogenesis, for example, but not limited to,
metabolic diseases such as obesity, as well as related disorders
such as eating disorder, cachexia, diabetes mellitus, hypertension,
coronary heart disease, hypercholesterolemia, dyslipidemia,
osteoarthritis, gallstones, and sleep apnea, and disorders related
to ROS defence, such as diabetes mellitus, neurodegenerative
disorders, and cancer, e.g, cancers of the reproductive organs, and
others, in cells, cell masses, organs and/or subjects.
14. Use of a nucleic acid molecule of the Mnk gene family or a
polynucleotide encoded thereby or a fragment or a variant of said
nucleic acid molecule or said polypeptide or an antibody, an
aptamer or another receptor recognizing a nucleic acid molecule of
the Mnk gene family or a polypeptide encoded thereby for
controlling the function of a gene and/or a gene product which is
influenced and/or modified by an Mnk homologous polypeptide.
15. Use of the nucleic acid molecule of the Mnk gene family or a
polynucleotide encoded thereby or a fragment or a variant of said I
nucleic acid molecule or said polypeptide or an antibody, an
aptamer or another receptor recognizing a nucleic acid molecule of
the Mnk gene family or a polypeptide encoded thereby for
identifying substances capable of interacting with an Mnk
homologous polypeptide.
16. A non-human transgenic animal exhibiting a modified expression
of an Mnk homologous polypeptide.
17. The animal of claim 16, wherein the expression of the Mnk
homologous polypeptide is increased and/or reduced.
18. A recombinant host cell exhibiting a modified expression of an
Mnk I homologous polypeptide.
19. The cell of claim 18 which is a human cell.
20. A method of identifying a (poly)peptide involved in the
regulation of energy homeostasis and/or metabolism of triglycerides
in a mammal comprising the steps of (a) contacting a collection of
(poly)peptides with an Mnk homologous polypeptide or a fragment
thereof under conditions that allow binding of said (poly)peptides;
(b) removing (poly)peptides which do not bind and (c) identifying
(poly)peptides that bind to said Mnk homologous polypeptide.
21. A method of screening for an agent which modulates the
interaction of an Mnk homologous polypeptide with a binding
target/agent, comprising the steps of (a) incubating a mixture
comprising (aa) an Mnk homologous polypeptide, or a fragment
thereof; (ab) a binding target/agent of said Mnk homologous
polypeptide or fragment thereof; and (ac) a candidate agent under
conditions whereby said Mnk polypeptide or fragment thereof
specifically binds to said binding target/agent at reference
affinity; (b) detecting the binding affinity of said Mnk
polypeptide or fragment thereof to said binding target to determine
an (candidate) agent-based affinity; and (c) determining a
difference between (candidate) agent-biased affinity and the
reference affinity.
22. A method of screening for an agent which modulates the activity
of an Mnk homologous polypeptide, comprising the steps of (a)
incubating a mixture comprising (aa) an Mnk homologous polypeptide,
or a fragment thereof; (ab) a candidate agent, under conditions
whereby said Mnk polypeptide or fragment thereof exhibits a
reference activity; (b) detecting the activity of said Mnk
polypeptide or fragment thereof to determine an (candidate)
agent-biased activity, and (c) determining a difference between a
(candidate) agent-biased activity and the reference activity.
23. The method of claim 21, wherein the candidate agent is selected
from peptides and low-molecular weight organic compounds.
24. The method of claim 20 wherein a known Mnk effector is used as
a positive control for assay development and/or validation of
candidate agents.
25. A method of producing a composition comprising mixing the
(poly)peptide identified by the method of claim 20 with a
pharmaceutically acceptable carrier, diluent and/or adjuvant.
26. The method of claim 25 wherein said composition is a
pharmaceutical composition for preventing, alleviating or treating
diseases and disorders related to body-weight regulation and
thermogenesis, for example, but not limited to, metabolic diseases
such as obesity, as well as related disorders such as eating
disorder, cachexia, diabetes mellitus, hypertension, coronary heart
disease, hypercholesterolemia, dyslipidemia, osteoarthritis,
gallstones, and sleep apnea, and disorders related to ROS defence,
such as diabetes mellitus, neurodegenerative disorders, and cancer,
e.g. cancers of the reproductive organs, and others, in cells, cell
masses, organs and/or subjects.
27. Use of a polypeptide as identified by the method of claim 20
for the preparation of a pharmaceutical composition for the
treatment, alleviation and/or prevention of diseases and disorders
related to body-weight regulation and thermogenesis, for example,
but not limited to, metabolic diseases such as obesity, as well as
related disorders such as eating disorder, cachexia, diabetes
mellitus, hypertension, coronary heart disease,
hypercholesterolemia, dyslipidemia, osteoarthritis, gallstones, and
sleep apnea, and disorders related to ROS defence, such as diabetes
mellitus, neurodegenerative disorders, and cancer, e.g. cancers of
the reproductive organs, and others, in cells, cell masses, organs
and/or subjects.
28. Use of a nucleic acid molecule of the Mnk family or of a
fragment thereof for the preparation of a non-human animal which
over or under-expresses the Mnk gene product.
29. Kit comprising at least one of (a) an Mnk nucleic acid molecule
or a fragment thereof; (b) a vector comprising the nucleic acid of
(a); (c) a host cell comprising the nucleic acid of (a) or the
vector of (d) a polypeptide encoded by the nucleic acid of (a); (e)
a fusion polypeptide encoded by the nucleic acid of (a) (f) an
antibody, an aptamer or another receptor against the nucleic acid
of (a) or the polypeptide of (d) or (e) and (g) an anti-sense
oligonucleotide of the nucleic acid of (a).
30. Use of an effector of an Mnk polypeptide for the manufacture of
an agent for the prophylaxis, treatment or diagnosis of diseases
and disorders related to body-weight regulation and thermogenesis,
for example, but not limited to, metabolic diseases such as
obesity, as well as related disorders such as eating disorder,
cachexia, diabetes mellitus, hypertension, coronary heart disease,
hypercholesterolemia, dyslipidemia, osteoarthritis, gallstones, and
sleep ) apnea, and disorders related to ROS defence, such as
diabetes mellitus, neurodegenerative disorders, and cancer, e.g.
cancers of the reproductive organs.
31. The use of claim 30, wherein the effector is of an Mnk2 or Mnk1
polypeptide.
32. The use of claim 30 wherein the effector is selected from
staurosporine or pyrazole derivatives.
33. The use of claim 30 wherein the effector is CGP57380 or a
derivative thereof.
Description
[0001] This invention relates to the use of nucleic acid sequences
of the MAP kinase-interacting kinase (Mnk) gene family and amino
acid sequences encoded thereby, and to the use of these sequences
or effectors of Mnk nucleic acids or polypeptides, particularly Mnk
kinase inhibitors and activators, in the diagnosis, study,
prevention, and treatment of diseases and disorders related to
body-weight regulation and thermogenesis, for example, but not
limited to, metabolic diseases such as obesity, as well as related
disorders such as eating disorder, cachexia, diabetes mellitus,
hypertension, coronary heart disease, hypercholesterolemia,
dyslipidemia, osteoarthritis, gallstones, and sleep apnea, and
disorders related to ROS defence, such as diabetes mellitus,
neurodegenerative disorders, and cancer, e.g. cancers of the
reproductive organs.
[0002] There are several metabolic diseases of human and animal
metabolism, eg., obesity and severe weight loss, that relate to
energy imbalance where caloric intake versus energy expenditure is
imbalanced. Obesity is one of the most prevalent metabolic disorder
in the world. It is a still poorly understood human disease that
becomes more and more relevant for western society. Obesity is
defined as a body weight more than 20% in excess of the ideal body
weight, frequently resulting in a significant impairment of health.
It is associated with an increased risk for cardiovascular disease,
hypertension, diabetes, hyperlipidemia and an increased mortality
rate. Besides severe risks of illness, individuals suffering from
obesity are often isolated socially.
[0003] Obesity is influenced by genetic, metabolic, biochemical,
psychological, and behavioral -factors. As such, it is a complex
disorder that must be addressed on several fronts to achieve
lasting positive clinical outcome. Since obesity is not to be
considered as a single disorder but as a heterogeneous group of
conditions with (potential) multiple causes, it is also
characterized by elevated fasting plasma insulin and an exaggerated
insulin response to oral glucose intake (Koltermann, J. Clin.
Invest 65, 1980, 1272-1284). A clear involvement of obesity in type
2 diabetes mellitus can be confirmed (Kopelman, Nature 404, 2000,
635-643).
[0004] The molecular factors regulating food intake and body weight
balance are incompletely understood. Even if several candidate
genes have been described which are supposed to influence the
homeostatic system(s) that regulate body mass/weight, like leptin,
VCPI, VCPL or the peroxisome proliferator-activated receptor-gamma
co-activator, the distinct molecular mechanisms and/or molecules
influencing obesity or body weight/body mass regulations are not
known. In addition, several single-gene mutations resulting in
obesity have been described in mice, implicating genetic factors in
the etiology of obesity (Friedman and Leibel, 1990, Cell 69:
217-220). In the obese mouse, a single gene mutation (obese)
results in profound obesity, which is accompanied by diabetes
(Friedman et. al., 1991, Genomics 11: 1054-1062).
[0005] Therefore, the technical problem underlying the present
invention was to provide for means and methods for modulating
(pathological) metabolic conditions influencing thermogenesis,
body-weight regulation and/or energy homeostatic circuits. The
solution to said technical problem is achieved by providing the
embodiments characterized in the claims.
[0006] Accordingly, the present invention relates to genes with
novel functions in body-weight regulation, energy homeostasis,
metabolism, and obesity. The present invention provides for a
specific gene involved in the regulation of diseases and disorders
related to body-weight regulation and thermogenesis, for example,
but not limited to, metabolic diseases such as obesity, as well as
related disorders such as eating disorder, cachexia, diabetes
mellitus, hypertension, coronary heart disease,
hypercholesterolemia, dyslipidemia, osteoarthritis, gallstones,
cancers of the reproductive organs, and sleep apnea, and disorders
related to ROS defence, such as diabetes mellitus,
neurodegenerative disorders, and cancer. The present invention
describes the human Mnk genes as being involved in those conditions
mentioned above, in particular the human Mnk2 gene variants.
[0007] The term "GenBank Accession number" relates to National
Center for Biotechnology Information (NCBI) GenBank database
entries (Benson et al, Nucleic Acids Res. 28, 2000, 15-18).
[0008] Protein kinases are important molecules involved in the
regulation of many cellular functions. The Drosophila melanogaster
LK6 serin/threonine kinase gene has been described as a short-lived
kinase that can associate with microtubules (J. Cell Sci. 1997
110(2):209-219). Genetic analysis in the development of the
Drosophila compound eye suggested a role in the modulation of the
RAS signalling pathway (Genetics 2000 156(3):1219-1230). As
described in this invention, the closest human homologues of
Drosophila LK6 kinase are the MAP kinase-interacting kinase 2
(Mnk2, for example the variants Mnk2a and Mnk2b) and MAP
kinase-interacting kinase 1 (Mnk1). All three proteins are
predominantly localized in the cytoplasm. Mnks are phosphorylated
by the pk42 MAP kinases Erk1 and Erk2 and the p38 MAP kinases. This
phophorylation is triggered in response to growth factors, phorbol
esters and oncogenes like Ras and Mos as well as by stress
signaling molecules and cytokines. The phosphorylation of Mnk
proteins stimulates its kinase activity towards eukaryotic
initiation factor 4E (EMBO J. 16: 1909-1920 (1997), Mol Cell Biol
19:1871-1880 (1999), Mol Cell Biol 21: 743-754 (2001)).
Phosphorylation of eukaryotic initiation factor 4E (eIF4E) results
in a regulation of protein translation (Mol Cell Biol 22: 5500-5511
(2001)).
[0009] There are different hypothesis describing the mode of
stimulation of the protein translation by Mnk proteins. Most
publications described a positive stimulatory effect on the
cap-dependent protein translation upon activation of MAP
kinase-interacting kinases. Thus, activation of Mnk proteins might
lead to an indirect stimulation or regulation of protein
translation, for example by the action on cytosolic phospholipase 2
alpha (BBA 1488:124-138, 2000).
[0010] Inhibitors of Mnk (referred to as CGP57380 and CGP052088)
were described in the prior art (see, Knauf et al., 2001, Mol.
Cell. Biol. 21:5500, Tschopp et al., 2000, Mol Cell Biol Res Comm
3:205 and Slentz-Kesler et al.,.2000, Genomics 69:63). CGP052088 is
a staurosporine derivative with an IC50 of 70 nM for inhibition of
in vitro kinase activity of Mnk1. CGP57380 is a selective
low-molecular weight, non cytotoxic inhibitor of Mnk2 (Mnk2a or
Mnk2b) or Mnk1. The addition of CGP57380 to cell culture cells
transfected with Mnk2 (Mnk2a or Mnk2b) or Mnk1 resulted in a strong
reduction in phosphorylated eIF4E.
[0011] So far, it has not been described that Mnk kinases are
involved in the regulation of body-weight and thermogenesis, and
thus may be associated with metabolic diseases such as obesity, as
well as related disorders such as eating disorder, cachexia,
diabetes mellitus, hypertension, coronary heart disease,
hypercholesterolemia, dyslipidemia, osteoarthritis, gallstones, and
sleep apnea, and disorders related to ROS defence, such as diabetes
mellitus, neurodegenerative disorders, and cancer, e.g. cancers of
the reproductive organs. In this application we demonstrate that
the correct gene doses of Mnk kinases are essential for maintenance
of energy homeostasis. A genetic screen was used to identify that
mutation of Mnk kinase homologous genes causes obesity, reflected
by a significant increase of triglyceride content, the major energy
storage substance. Furthermore, in this invention we relate to
mutations of Mnk kinases that affect the activity of uncoupling
proteins (UCPs), thereby leading to an altered mitochondrial
activity. We also relate to the treatment of metabolic disorders
with the Mnk-specific inhibitor CGP57380 and derivatives
thereof.
[0012] In this invention we demonstrate that the correct gene dose
of the Drosophila melanogaster homologue of Mnk is essential for
maintenance of energy homeostasis in adult flies and for the
activity of mitochondrial uncoupling protein. A genetic screen was
used to identify that mutation of an. Mnk homologous gene causes
obesity in Drosophila melanogaster, reflected by a significant
increase of triglyceride content, the major energy storage
substance. In a second screen designed to identify factors that
modulate activity of uncoupling protein, we discovered that
mutation of this Mnk homologous gene caused a reduction the
activity of uncoupling protein. Thus, the invention is also based
on the finding that the Drosophila homologue of Mnk is contributing
to membrane stability and/or function of organelles, preferably
mitochondria. It was found that mutations in LK6 kinases affect the
activity of uncoupling proteins (UCPs), thereby leading to an
altered mitochondrial activity.
[0013] Further, we show that the mouse homologue of the Mnk2 gene
is regulated by fasting arid by genetically induced obesity.
Furthermore, the Mnk2 mRNA is strongly upregulated during adipocyte
differentiation in vitro (see EXAMPLES). This invention shows that
Mnk2 transcripts are expressed in most mouse tissues but with
highest expression levels in white (WAT) and brown adipose tissue
(BAT). The expression in white adipose tissue is reduced by approx.
60% in fasted mice and in ob/ob mice.
[0014] The analysis of actin-mMnk2DN transgenic mice showed that
the ectopic expression of mMnk2DN transgene (see Examples) leads to
an clear increase in bodyweight. The effect seems to be
diet-independent, as it can be seen on control diet as well as on
high fat diet. Thus, we conclude that Mnk2 is playing an important
role in the regulation of body-weight.
[0015] In addition, we found that the relative expression levels of
both human Mnk2 splice variants is the same for all tissues
analysed. Both Mnk2 variants show highest expression levels in
human tissues relevant for metabolic disorders namely adipose and
muscle tissue. Furthermore, both Mnk2 variants are upregulated
during human adipocyte differentiation. Thus, we conclude that Mnk2
(or variants thereof) has a function in the metabolism of mature
human adipocytes.
[0016] We also found that cellular triglyceride levels in Mnk2
overexpressing cells were significantly lower from day 4 to day 12
of adipogenesis compared to that in the control cells. Furthermore,
Mnk2 overexpressing cells were less effective at synthesising
lipids from exogenous glucose. Consequently, the levels of insulin
stimulated lipid synthesis are significantly lower at day 12 of
adipogenesis when compared to control cells. We also found that
transport of exogenous fatty acids across the plasma membrane of
Mnk2 overexpressing cells and hence esterification of these
metabolites were considerably lower at day 12 of adipogenesis when
compared to control cells.
[0017] Polynucleotides encoding a protein with homologies to
proteins of the Mnk kinase family are suitable to investigate
diseases and disorders as described above. Discovery of molecules
related to Mnk kinases satisfies a need in the art by providing new
compositions useful in diagnosis, treatment, and prognosis of
diseases and disorders as described above.
[0018] Before the present proteins, nucleotide sequences, and
methods are described, it is understood that this invention is not
limited to the particular methodology, protocols, cell lines,
vectors, and reagents described as these may vary. It is also to be
understood that the terminology used herein is for the purpose of
describing particular embodiments only, and is not intended to
limit the scope of the present invention, which will be limited
only by the appended claims. Unless defined otherwise, all
technical and scientific terms used herein have the same meanings
as commonly understood by one of ordinary skill in the art to which
this invention belongs. Although any methods and materials similar
or equivalent to those described herein can be used in the practice
or testing of the present invention, the preferred methods,
devices, and materials are now described. All publications
mentioned herein are incorporated herein by reference for the
purpose of describing and disclosing the cell lines, vectors, and
methodologies, which are reported in the publications which might
be used in connection with the invention. Nothing herein is to be
construed as an admission that the invention is not entitled to
antedate such disclosure.
[0019] The present invention discloses that Mnk homologous proteins
are regulating the energy homeostasis and fat metabolism,
especially the metabolism and storage of triglycerides, and
polynucleotides, which identify and encode the proteins disclosed
in this invention. The present invention also discloses that Mnk
homologous proteins are directly or indirectly involved in membrane
stability and/or function of organelles, in particular
mitochondria, and polynucleotides, which identify and encode the
proteins disclosed in this invention. The invention also relates to
vectors, host cells, antibodies, and recombinant methods for
producing the polypeptides and polynucleotides of the invention.
The invention also relates to the use of these sequences in the
diagnosis, study, prevention, and treatment of diseases and
disorders related to body-weight regulation and thermogenesis, for
example, but not limited to, metabolic diseases such as obesity, as
well as related disorders such as eating disorder, cachexia,
diabetes mellitus, hypertension, coronary heart disease,
hypercholesterolemia, dyslipidemia, osteoarthritis, gallstones, and
sleep apnea, and disorders related to ROS defence, such as diabetes
mellitus, neurodegenerative disorders, and cancer, e.g. cancers of
the reproductive organs.
[0020] Mnk homologous proteins and nucleic acid molecules coding
therefore are obtainable from insect or vertebrate species, e.g.
mammals or birds. Particularly preferred are human Mnk homologous
polypeptides and nucleic acids encoding such polypeptides,
particularly polypeptides and nucleic acids encoding a human Mnk2
protein (splice variant Mnk2a, Genbank Accession No. AF237775 as
shown in FIGS. 3D and 3E, or splice variant Mnk2b, GenBank
Accession AF237776 or No. NM.sub.--017572.1, as shown in ) FIGS. 3F
and 3G, Genbank Accession No. AF237775 is identical to formerly
Genbank Accession No. XM.sub.--030637 which was removed at the
submitters request; see a Clustal W multiple sequence alignment in
FIG. 3B, see also sequences in FIGS. 3D-G) or a human Mnk1 protein
(Genbank Accession No. AB000409.1 and NM.sub.--003684.2 as shown in
FIGS. 3H and 3I); Genbank Accession No. AB000409 is identical to
formerly Genbank Accession No. XM.sub.--001 600 which was removed
at the submitters request; see a Clustal W multiple sequence
alignment in FIG. 3C).
[0021] The invention particularly relates to a nucleic acid
molecule encoding a polypeptide contributing to regulating the
energy homeostasis and the metabolism of triglycerides, and/or
contributing to membrane stability and/or function of organelles,
wherein said nucleic acid molecule comprises
[0022] (a) the nucleotide sequences of Genbank Accession Nos.
AF237775, NM.sub.--017572.1, AB000409.1,. or NM.sub.--003684.2,
and/or the complement thereof,
[0023] (b) a nucleotide sequence which hybridizes at 50.degree. C.
in a solution containing 1.times.SSC and 0.1% SDS to the nucleic
acid molecule of (a), particularly a nucleic acid encoding the
amino acid sequences as shown in FIG. 3,
[0024] (c) a sequence corresponding to the sequences of (a) or (b)
within the degeneration of the genetic code,
[0025] (d) a sequence which encodes a polypeptide which is at least
85%, preferably at least 90%, more preferably at least 95%, more
preferably at least 98% and up to 99,6% identical to the amino acid
sequences shown in FIG. 3,
[0026] (e) a sequence Which differs from the nucleic acid molecule
of (a) to (d) by mutation and wherein said mutation causes an
alteration, deletion, duplication or premature stop in the encoded
polypeptide or
[0027] (f) a partial sequence of any of the nucleotide sequences of
(a) to (e) having a length of at least 15 bases, preferably at
least 20 bases, more preferably at least 25 bases and most
preferably at least 50 bases.
[0028] The invention is based on the finding that Mnk homologous
proteins (herein referred to as Mnk), particularly Mnk2 (Mnk2a or
Mnk2b) or Mnk1, and the polynucleotides encoding these, are
involved in the regulation of triglyceride storage and therefore
energy homeostasis. The present invention also discloses that Mnk
homologous proteins are directly or indirectly involved in membrane
stability and/or function of organelles, in particular
mitochondria, and polynucleotides, which identify and encode the
proteins disclosed in this invention. The invention describes the
use of compositions comprising the nucleotides, proteins or
effectors thereof, e.g. antibodies, aptamers, anti-sense molecules,
ribozymes, RNAi molecules, peptides, low-molecular weight organic
molecules and other receptors recognizing the nucleic acid molecule
or the polypeptide, for the diagnosis, study, prevention, or
treatment of diseases and disorders related to body-weight
regulation and thermogenesis, for example, but not limited to,
metabolic diseases such as obesity, as well as related disorders
such as eating disorder, cachexia, diabetes mellitus, hypertension,
coronary heart disease, hypercholesterolemia, dyslipidemia,
osteoarthritis, gallstones, and sleep apnea, and disorders related
to ROS defence, such as diabetes mellitus, neurodegenerative
disorders, and cancer, e.g. cancers of the reproductive organs.
[0029] Accordingly, the present invention relates to genes with
novel functions in body-weight regulation, energy homeostasis,
metabolism, and obesity. To find genes with novel functions in
energy homeostasis, metabolism, and obesity, a functional genetic
screen was performed with the model organism Drosophila
melanogaster (Meigen). Drosophila melanogaster is one of the most
intensively studied organisms in biology and serves as a model
system for the investigation of many developmental and cellular
processes common to higher eukaryotes, including humans (see, for
example, Adams et al., Science 287: 2185-2195 (2000)). The success
of Drosophila melanogaster as a model organism is largely due to
the power of forward genetic screens to identify the genes that are
involved in a biological process (see, Johnston Nat Rev Genet 3:
176-188 (2002); Rorth, Proc Natl Acad Sci USA 93: 12418-12422
(1996)). One resource for screening was a proprietary Drosophila
melanogaster stock collection of EP-lines. The P-vector of this
collection has Gal4-UAS-binding sites fused to a basal promoter
that can transcribe adjacent genomic Drosophila sequences upon
binding of Gal4 to UAS-sites. This enables the EP-line collection
for overexpression of endogenous flanking gene sequences. In
addition, without activation of the UAS-sites, integration of the
EP-element into the gene is likely to cause a reduction of gene
activity, and allows determining its function by evaluating the
loss-of-function phenotype.
[0030] Triglycerides are the most efficient storage for energy in
cells, and are significantly increased in obese patients. In this
invention, we have used a genetic screen to identify, that
mutations of Lk6 homologous genes cause changes in the body weight
which is reflected by a significant change in the triglyceride
levels. In order to isolate genes with a function in energy
homeostasis, several thousand EP-lines were tested for their
triglyceride content after a prolonged feeding period. Lines with
significantly changed triglyceride content were selected as
positive candidates for further analysis. In this invention, the
content of triglycerides of a pool of flies with the same genotype
after feeding for six days was analyzed using a triglyceride assay,
as, for example, but not for limiting the scope of the invention,
is described below in the examples section. The change of
triglyceride content due to the loss of a gene function suggests
gene activities in energy homeostasis in a dose dependent manner
that controls the amount of energy stored as triglycerides.
[0031] The result of the triglyceride content analysis is shown in
FIG. 1. Flies homozygous for EP(3)3333 and EP(3)3576 integrations
were analyzed in the triglyceride assay. The average increase of
triglyceride content of the homozygous viable lines EP(3)3333 and
EP(3)3576 is approx. 140% (FIG. 1). Therefore, the very likely loss
of a gene activity in the gene locus 86F7 (estimated, chromosomal
localisation where the EP-vector of EP(3)3333 and EP(3)3576 flies
is integrated) is responsible for changes in the metabolism of the
energy storage triglycerides, therefore representing in both cases
an obese fly model. The increase of triglyceride content due to the
loss of a gene function suggests gene activities in energy
homeostasis in a dose dependent manner that controls the amount of
energy stored as triglycerides.
[0032] Nucleic acids encoding the Mnk protein of the present
invention were identified using a plasmid-rescue technique. Genomic
DNA sequences were isolated that are localised directly 3' to the
EP(3)3333 and EP(3)3576 integrations. Using those isolated genomic
sequences public databases like Berkeley Drosophila Genome Project
(GadFly; see also FlyBase (1 999) Nucleic Acids Research 27:85-88)
were screened thereby confirming the integration side of EP(3)3333
in the 5' region of a 5' exon of the Mnk homologous gene and
EP(3)3576 in the 5' region of an alternative 5' exon (FIG. 2). FIG.
2 shows the molecular organisation of this locus. Genomic DNA
sequence is represented by the assembly as a black dotted line in
the middle that includes the integration site of EP(3)3333 and
EP(3)3576. Numbers represent the coordinates of the genomic DNA
(starting at position 7544500 on chromosome 3R). Grey bars on the
two "cDNA"-lines represent the predicted genes (GadFly &
Magpie), and grey symbols on the "P-Elements"-line the EP-vector
integration sites. Predicted exons of gene CG17342 are shown as
dark grey bars and predicted introns as light grey bars.
[0033] Lk6 (the Mnk homologous gene in Drosophila) encodes for a
gene that is predicted by GadFly sequence analysis programs (GadFly
Accession Number CG17342). No functional data described the
regulation of obesity and metabolic diseases are available in the
prior art for the genes shown in FIG. 3, referred to as Mnk in the
present invention.
[0034] It is also preferred that the nucleic acid molecule encodes
a polypeptide contributing to membrane stability and/or function of
orgnelles and represents a protein of Drosophila which has been
found to be able to modify UCPs, see also appended examples. As
demonstrated in the appended examples, the here described
polypeptide (and encoding nucleic acid molecule) was able to
modify, e.g. enhance a specific eye phenotype in Drosophila which
was due to the overexpression of the Drosophila melanogaster gene
dUCPy. The overexpression of dUCPy (with homology to human UCPs) in
the compound eye of Drosophila led to a clearly visible eye defect
which can be used as a "read-out" for a genetical "modifier
screen".
[0035] In said "modifier screen" thousands of different genes are
mutagenized to modify their expression in the eye. Should one of
the mutagenized genes interact with dUCPy and modify its activity
an enhancement or suppression of the eye defect will occur. Since
such flies are easily to discern they can be selected to isolate
the interacting gene. As shown in the appended examples, a gene was
deduced that can enhance the eye defect induced by the activity of
dUCPy. This gene is called the LK6 gene of Drosophila with high
homologies to the human Mnk proteins, as described above. It is
envisaged that mutations in the herein described Mnk-polypeptides
(and genes) lead to phenotypic and/or physiological chances which
may comprise a modified and altered mitochondrial activity. This,
in turn, may lead to, inter alia, an altered energy metabolism,
altered thermogenesis and/or altered energy homeostasis. As shown
in the appended examples, a gene was deduced that can enhance the
eye defect induced by the activity of dUCPy.
[0036] Mnk homologous proteins and nucleic acid molecules coding
therefor are obtainable from insect or vertebrate species, e.g.
mammals or birds. Particularly preferred are nucleic acids encoding
the human Lk6/Mnk homologs, particularly Mnk2 variants (Mnk2a or
Mnk2b) or Mnk1. The present invention is describing a polypeptide
comprising the amino acid sequence of Mnk, particularly Mnk2
variants (Mnk2a or Mnk2b) or Mnk1. A comparison (Clustal X 1.8)
between the Mnk proteins of different species (human and
Drosophila) was conducted and is shown in FIG. 3A. Based upon
homology, Mnk protein of the invention and each homologous protein
or peptide may share at least some activity.
[0037] In a particular embodiment, the invention encompasses the
polynucleotide comprising the nucleic acid sequence of GenBank
Accession Number AF237775, NM.sub.--017572.1, AB000409.1, or
NM.sub.--003684.2. It will be appreciated by those skilled in the
art that as a result of the degeneracy of the genetic code, a
multitude of nucleotide sequences encoding Mnk, some bearing
minimal homology to the nucleotide sequences of any known and
naturally occurring gene, may be produced. Thus, the invention
contemplates each and every possible variation of nucleotide
sequence that could be made by selecting combinations based on
possible codon choices.
[0038] These combinations are made in accordance with the standard
triplet genetic code as applied to the nucleotide sequences of
naturally occurring Mnk, and all such variations are to be
considered as being specifically disclosed. Although nucleotide
sequences which encode Mnk and its variants are preferably capable
of hybridising to the nucleotide sequences of the naturally
occurring Mnk under appropriately selected conditions of
stringency, it may be advantageous to produce nucleotide sequences
encoding Mnk or its derivatives possessing a substantially
different codon usage. Codons may be selected to increase the rate
at which expression of the peptide occurs in a particular
prokaryotic or eukaryotic host in accordance with the frequency
with which particular codons are utilised by the host. Other
reasons for substantially altering the nucleotide sequence encoding
Mnk and its derivatives without altering the encoded amino acid
sequences include the production of RNA transcripts having more
desirable properties, such as a greater half-life, than transcripts
produced from the naturally occurring sequences. The invention also
encompasses production of DNA sequences, or portions thereof, which
encode Mnk and its derivatives, entirely by synthetic chemistry.
After production, the synthetic sequence may be inserted into any
of the many available expression vectors and cell systems using
reagents that are well known in the art at the time of the filing
of this application. Moreover, synthetic chemistry may be used to
introduce mutations into a sequence encoding Mnk any portion
thereof.
[0039] Also encompassed by the invention are polynucleotide
sequences that are capable of hybridizing to the claimed nucleotide
sequences, and in particular, those shown in GenBank Accession
Numbers AF237775, NM.sub.--017572.1, AB000409.1, or
NM.sub.--003684.2, under various conditions of stringency.
Hybridization conditions are based on the melting temperature (Tm)
of the nucleic acid binding complex or probe, as taught in Wahl, G.
M. and S. L. Berger (1987: Methods Enzymol. 152:399-407) and
Kimmel, A. R. (1987; Methods Enzymol. 152:507-511), and may be used
at a defined stringency. Preferably, hybridization under stringent
conditions means that after washing for 1 h with 1.times.SSC and
0.1% SDS at 50.degree. C., preferably at 55.degree. C., more
preferably at 62.degree. C. and most preferably at 68.degree. C.,
particularly for 1 h in 0.2.times.SSC and 0.1% SDS at 50.degree.
C., preferably at 55.degree. C., more preferably at 62.degree. C.
and most preferably at 68.degree. C., a positive hybridization
signal is observed. Altered nucleic acid sequences encoding Mnk
which are encompassed by the invention include deletions,
insertions, or substitutions of different nucleotides resulting in
a polynucleotide that encodes the same or a functionally equivalent
Mnk.
[0040] The encoded proteins may also contain deletions, insertions,
or substitutions of amino acid residues, which produce a silent
change and result in a functionally equivalent Mnk. Deliberate
amino acid substitutions may be made on the basis of similarity in
polarity, charge, solubility, hydrophobicity, hydrophilicity,
and/or the amphipathic nature of the residues as long as the
biological activity of Mnk is retained. For example, negatively
charged amino acids may include aspartic acid and glutamic acid;
positively charged amino acids may include lysine and arginine; and
amino acids with uncharged polar head groups having similar
hydrophilicity values may include leucine, isoleucine, and valine;
glycine and alanine; asparagine and glutamine; serine and
threonine; phenylalanine and tyrosine.
[0041] Also included within the scope of the present invention are
alleles of the genes encoding Mnk. As used herein, an "allele" or
"allelic sequence" is an alternative form of the gene, which may
result from at least one mutation in the nucleic acid sequence.
Alleles may result in altered mRNAs or polypeptides whose
structures or function may or may not be altered. Any given gene
may have none, one, or many allelic forms. Common mutational
changes, which give rise to alleles, are generally ascribed to
natural deletions, additions, or substitutions of nucleotides. Each
of these types of changes may occur alone, or in combination with
the others, one or more times in a given sequence. Methods for DNA
sequencing which are well known and generally available in the art
may be used to practice any embodiments of the invention. The
methods may employ such enzymes as the Klenow fragment of DNA
polymerase I, SEQUENASE DNA Polymerase (US Biochemical Corp,
Cleveland Ohio), Taq polymerase (Perkin Elmer), thermostable T7
polymerase (Amersham, Chicago, Ill.), or combinations of
recombinant polymerases and proof-reading exonucleases such as the
ELONGASE Amplification System (GIBCO/BRL, Gaithersburg, Md.).
Preferably, the process is automated with machines such as the
Hamilton MICROLAB 2200 (Hamilton, Reno Nev.), Peltier thermal
cycler (PTC200; MJ Research, Watertown, Mass.) and the ABI 377 DNA
sequencers (Perkin Elmer). The nucleic acid sequences encoding Mnk
may be extended utilising a partial nucleotide sequence and
employing various methods known in the art to detect upstream
sequences such as promoters and regulatory elements. For example,
one method which may be employed, "restriction-site" PCR, uses
universal primers to retrieve unknown sequence adjacent to a known
locus (Sarkar, G. (1993) PCR Methods Applic. 2:318-322). Inverse
PCR may also be used to amplify or extend sequences using divergent
primers based on a known region (Triglia, T. et al. (1988) Nucleic
Acids Res. 16:8186). Another method which may be used is capture
PCR which involves PCR amplification of DNA fragments adjacent to a
known sequence in human and yeast artificial chromosome DNA
(Lagerstrom, M. et al. (PCR Methods Applic. 1:111-119). Another
method which may be used to retrieve unknown sequences is that of
Parker, J. D. et al. (1 991; Nucleic Acids Res. 19:3055-3060).
Additionally, one may use PCR, nested primers, and PROMOTERFINDER
libraries to walk in genomic DNA (Clontech, Palo Alto, Calif.).
This process avoids the need to screen libraries and is useful in
finding intron/exon junctions.
[0042] When screening for full-length cDNAs, it is preferable to
use libraries that have been size-selected to include larger cDNAs.
Also, random-primed libraries are preferable, in that they will
contain more sequences, which contain the 5' regions of genes. Use
of a randomly primed library may be especially preferable for
situations in which an oligo d(T) library does not yield a
full-length cDNA. Genomic libraries may be useful for extension of
sequence into the 5' and 3' non-transcribed regulatory regions.
Capillary electrophoresis systems, which are commercially
available, may be used to analyse the size or confirm the
nucleotide sequence of sequencing or PCR products. In particular,
capillary sequencing may employ flowable polymers for
electrophoretic separation, four different fluorescent dyes (one
for each nucleotide) which are laser activated, and detection of
the emitted wavelengths by a charge coupled devise camera.
Output/light intensity may be converted to electrical signal using
appropriate software (e.g. GENOTYPER and SEQUENCE NAVIGATOR, Perkin
Elmer) and the entire process from loading of samples to computer
analysis and electronic data display may be computer controlled.
Capillary electrophoresis is especially preferable for the
sequencing of small pieces of DNA, which might be present in
limited amounts in a particular sample.
[0043] In another embodiment of the invention, polynucleotide
sequences or fragments thereof which encode Mnk, or fusion proteins
or functional equivalents thereof, may be used in recombinant DNA
molecules to direct expression of Mnk in appropriate host cells.
Due to the inherent degeneracy of the genetic code, other DNA
sequences, which encode substantially the same, or a functionally
equivalent amino acid sequence may be produced and these sequences
may be used to clone and express Mnk. As will be understood by
those of skill in the art, it may be advantageous to produce
Mnk-encoding nucleotide sequences possessing non-naturally
occurring codons. For example, codons preferred by a particular
prokaryotic or eukaryotic host can be selected to increase the rate
of protein expression or to produce a recombinant RNA transcript
having desirable properties, such as a half-life, which is longer
than that of a transcript generated from the naturally occurring
sequence. The nucleotide sequences of the present invention can be
engineered using methods generally known in the art in order to
alter Mnk encoding sequences for a variety of reasons, including
but not limited to, alterations which modify the cloning,
processing, and/or expression of the gene product. DNA shuffling by
random fragmentation and PCR reassembly of gene fragments and
synthetic oligonucleotides may be used to engineer the nucleotide
sequences. For example, site-directed mutagenesis may be used to
insert new restriction sites, alter glycosylation patterns, change
codon preference, produce splice variants, or introduce mutations,
and so forth.
[0044] In another embodiment of the invention, natural, modified,
or recombinant nucleic acid sequences encoding Mnk may be ligated
to a heterologous sequence to encode a fusion protein. For example,
to screen peptide libraries for inhibitors of Mnk activities, it
may be useful to construct chimeric Mnk proteins that can be
recognised by a commercially available antibodies. A fusion protein
may also be engineered to contain a cleavage site located between
the Mnk encoding sequence and the heterologous protein sequences,
so that Mnk may be cleaved and purified away from the heterologous
moiety. In another embodiment, sequences encoding Mnk may be
synthesised, in whole or in part, using chemical methods well known
in the art (see Caruthers et al. (1980) Nucl. Acids Res. Symp. Ser.
7:215-223, Horn et al. (1980) Nucl. Acids Res. Symp. Ser.
7:225-232). Alternatively, the proteins themselves may be produced
using chemical methods to synthesise the amino acid sequence of
Mnk, or a portion thereof. For example, peptide synthesis can be
performed using various solid-phase techniques (Roberge et al.
(1995) Science 269:202-204) and automated synthesis may be
achieved, for example, using the ABI 431A peptide synthesiser
(Perkin Elmer). The newly synthesised peptide may be substantially
purified by preparative high performance liquid chromatography
(e.g., Creighton, T. (1983) Proteins, Structures and Molecular
Principles, WH Freeman and Co., New York, N.Y.). The composition of
the synthetic peptides may be confirmed by amino acid analysis or
sequencing (e.g., the Edman degradation procedure; Creighton,
supra). Additionally, the amino acid sequences of Mnk, or any part
thereof, may be altered during direct synthesis and/or combined
using chemical methods with sequences from other proteins, or any
part thereof, to produce a variant polypeptide.
[0045] In order to express a biologically active Mnk, the
nucleotide sequences encoding Mnk functional equivalents, may be
inserted into appropriate expression vectors, i.e., a vector, which
contains the necessary elements for the transcription and
translation of the inserted coding sequence. Methods, which are
well known to those skilled in the art, may be used to construct
expression vectors containing sequences encoding Mnk and
appropriate transcriptional and translational control elements.
These methods include in vitro recombinant DNA techniques.
synthetic techniques, and in vivo genetic recombination. Such
techniques are described in Sambrook, J. et al. (1989) Molecular
Cloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview,
N.Y., and Ausubel, F. M. et al. (1989) Current Protocols in
Molecular Biology, John Wiley & Sons, New York, N.Y.
[0046] Regulatory elements include for example a promoter, an
initiation codon, a stop codon, a mRNA stability regulatory
element, and a polyadenylation signal. Expression of a
polynucleotide can be assured by (i) constitutive promoters such as
the Cytomegalovirus (CMV) promoter/enhancer region, (ii) tissue
specific promoters such as the insulin promoter (see, Soria et al.,
2000, Diabetes 49:157), SOX2 gene promotor (see Li et al., 1998,
Curr. Biol. 8:971-4), Msi-1 promotor (see Sakakibara et al., 1997,
J. Neuroscience 17:8300-8312), alpha-cardia myosin heavy chain
promotor or human atrial natriuretic factor promotor (Klug et al.,
1996, J. clin. Invest 98:216-24; Wu et al., 1989, J. Biol. Chem.
264:6472-79)or (iii) inducible promoters such as the tetracycline
inducible system. Expression vectors can also contain a selection
agent or marker gene that confers antibiotic resistance such as the
neomycin, hygromycin or puromycin resistance genes. These methods
include in vitro recombinant DNA techniques, synthetic techniques,
and in vivo genetic recombination. Such techniques are described in
Sambrook, J. et al. (1989) Molecular Cloning, A Laboratory Manual,
Cold Spring Harbor Press, Plainview, N.Y. and Ausubel, F. M. et al.
(1989) Current Protocols in Molecular Biology, John Wiley &
Sons, New York, N.Y. In a further embodiment of the invention,
natural, modified or recombinant nucleic acid sequences encoding
the proteins of the invention and homologous proteins may be
ligated to a heterologous sequence to encode a fusion protein.
[0047] A variety of expression vector/host systems may be utilized
to contain and express sequences encoding the proteins or fusion
proteins. These include, but are not limited to, micro-organisms
such as bacteria transformed with recombinant bacteriophage,
plasmid or cosmid DNA expression vectors; yeast transformed with
yeast expression vectors; insect cell systems infected with virus
expression vectors (e.g., baculovirus, adenovirus, adeno-associated
virus, lentiverus, retrovirus); plant cell systems transformed with
virus expression vectors (e.g., cauliflower mosaic virus, CaMV;
tobacco mosaic virus, TMV) or with bacterial expression vectors
(e.g., Ti or PBR322 plasmids); or animal cell systems.
[0048] The "control elements" or "regulatory sequences" are those
non-translated regions of the vectors, e.g. enhancers, promoters,
5' and 3' untranslated regions, which interact with host cellular
proteins to carry out transcription and translation. Such elements
may vary in their strength and specificity. Depending on the vector
system and host utilised, any number of suitable transcription and
translation elements, including constitutive and inducible
promoters, may be used. For example, when cloning in bacterial
systems, inducible promoters such as the hybrid lacZ promoter of
the BLUESCRIPT phagemid (Stratagene, LaJolla, Calif.) or PSPORT1
plasmid (Gibco BRL) and the like may be used. The baculovirus
polyhedrin promoter may be used in insect cells. Promoters and
enhancers derived from the genomes of plant cells (e.g., heat
shock, RUBISCO; and storage protein genes) or from plant viruses
(e.g., viral promoters and leader sequences) may be cloned into the
vector. In mammalian cell systems, promoters from mammalian genes
or from mammalian viruses are preferable. If it is necessary to
generate a cell line that contains multiple copies of the sequences
encoding Mnk, vectors based on SV40 or EBV may be used with an
appropriate selectable marker.
[0049] In bacterial systems, a number of expression vectors may be
selected depending upon the use intended for Mnk. For example, when
large quantities of Mnk are needed for the induction of antibodies,
vectors, which direct high level expression of fusion proteins that
are readily purified, may be used. Such vectors include, but are
not limited to, the multifunctional E. coli cloning and expression
vectors such as the BLUESCRIPT phagemid (Stratagene), in which the
sequence encoding Mnk may be ligated into the vector in frame with
sequences for the amino-terminal Met and the subsequent 7 residues
of .beta.-galactosidase so that a hybrid protein is produced; pIN
vectors (Van Heeke, G. and S. M. Schuster (1989) J. Biol. Chem.
264:5503-5509); and the like. Vectors of the pGEX series (Amersham
Biosciencies, Uppsala, Sweden) may also be used to express foreign
polypeptides as fusion proteins with Glutathione S-Transferase
(GST). In general, such fusion proteins are soluble and can easily
be purified from lysed cells by adsorption to glutathione-agarose
beads followed by elution in the presence of free glutathione.
Proteins made in such systems may be designed to include heparin,
thrombin, or factor XA protease cleavage sites so that the cloned
polypeptide of interest can be released from the GST moiety at
will. In the yeast, Saccharomyces cerevisiae, a number of vectors
containing constitutive or inducible promoters such as alpha
factor, alcohol oxidase, and PGH may be used. For reviews, see
Ausubel et al., (supra) and Grant et al. (1987) Methods Enzymol.
153:516-544.
[0050] In cases where plant expression vectors are used, the
expression of sequences encoding Mnk may be driven by any of a
number of promoters. For example, viral promoters such as the 35S
and 19S promoters of CaMV may be used alone or in combination with
the omega leader sequence from TMV (Takamatsu, N. (1987) EMBO J.
6:307-311). Alternatively, plant promoters such as the small
subunit of RUBISCO or heat shock promoters may be used (Coruzzi, G.
et al. (1984) EMBO J. 3:1671-1680; Broglie, R. et al. (1984)
Science 224:838-843; and Winter, J. et al. (1991) Results Probl.
Cell Differ. 17:85-105). These constructs can be introduced into
plant cells by direct DNA transformation or pathogen-mediated
transfection. Such techniques are described in a number of
generally available reviews (see, for example, Hobbs, S. or Murry,
L. E. in McGraw Hill Yearbook of Science and Technology (1992)
McGraw Hill, New York, N.Y.; pp. 191-196).
[0051] An insect system may also be used to express Mnk. For
example, in one such system, Autographa californica nuclear
polyhedrosis virus (AcNPV) is used as a vector to express foreign
genes in Spodoptera frugiperda cells or in Trichoplusia larvae. The
sequences encoding Mnk may be cloned into a non-essential region of
the virus, such as the polyhedrin gene, and place under control of
the polyhedrin promoter. Successful insertions of Mnk will render
the polyhedrin gene inactive and produce recombinant virus lacking
coat protein. The recombinant viruses may then be used to infect,
for example, S. frugiperda cells of Trichoplusia larvae in which
Mnk may be expressed (Engelhard, E. K. et al. (1994) Proc. Nat.
Acad. Sci. 91:3224-3227).
[0052] In mammalian host cells, a number of viral-based expression
systems may be utilised. In cases where an adenovirus is used as an
expression vector, sequences encoding Mnk may be ligated into an
adenovirus transcription/translation complex consisting of the late
promoter and tripartite leader sequence. Insertion in a
non-essential E1 or E3 region of the viral genome may be used to
obtain viable viruses which are capable of expressing Mnk in
infected host cells (Logan, J. and Shenk, T. (1984) Proc. Natl.
Acad. Sci. 81:3655-3659). In addition, transcription enhancers,
such as the Rous sarcoma virus (RSV) enhancer, may be used to
increase expression in mammalian host cells.
[0053] Specific initiation signals may also be used to achieve more
efficient translation of sequences encoding Mnk. Such signals
include the ATG initiation codon and adjacent sequences. In cases
where sequences encoding Mnk, its initiation codons, and upstream
sequences are inserted into the appropriate expression vector, no
additional transcriptional or translational control signals may be
needed. However, in cases where only coding sequence, or a portion
thereof, is inserted, exogenous translational control signals
including the ATG initiation codon should be provided. Furthermore,
the initiation codon should be in the correct reading frame to
ensure translation of the entire insert. Exogenous translational
elements and initiation codons may be of various origins, both
natural and synthetic. The efficiency of expression may be enhanced
by the inclusion of enhancers which are appropriate for the
particular cell system which is used, such as those described in
the literature (Scharf, D. et al. (1994) Results Probl. Cell
Differ. 20:125-162).
[0054] In addition, a host cell strain may be chosen for its
ability to modulate the expression of the inserted sequences or to
process the expressed protein in the desired fashion. Such
modifications of the polypeptide include, but are not limited to,
acetylation, carboxylation, glycosylation, phosphorylation,
lipidation, and acylation. Post-translational processing which
cleaves a "prepro" form of the protein may also be used to
facilitate correct insertion, folding and/or function. Different
host cells such as CHO, HeLa, MDCK, HEK293, and WI38, which have
specific cellular machinery and characteristic mechanisms for such
post-translational activities, may be chosen to ensure the correct
modification and processing of the foreign protein.
[0055] For long-term, high-yield production of recombinant
proteins, stable expression is preferred. For example, cell lines,
which stably express Mnk may be transformed using expression
vectors which may contain viral origins of replication and/or
endogenous expression elements and a selectable marker gene on the
same or on a separate vector. Following the introduction of the
vector, cells may be allowed to grow for 1-2 days in an enriched
media before they are switched to selective media. The purpose of
the selectable marker is to confer resistance to selection, and its
presence allows growth and recovery of cells, which successfully
express the introduced sequences. Resistant clones of stably
transformed cells may be proliferated using tissue culture
techniques appropriate to the cell type; Any number of selection
systems may be used to recover transformed cell lines. These
include, but are not limited to, the herpes simplex virus thymidine
kinase (Wigler, M. et al. (1977) Cell 11:223-32) and adenine
phosphoribosyltransferase (Lowy, I. et al. (1980) Cell 22:817-23)
genes, which can be employed in tk.sup.- or aprt.sup.-cells,
respectively. Also, antimetabolite, antibiotic or herbicide
resistance can be used as the basis for selection; for example,
dhfr which confers resistance to methotrexate (Wigler, M. et al.
(1980) Proc. Natl. Acad. Sci. 77:3567-70); npt, which confers
resistance to the aminoglycosides neomycin and G-418
(Colbere-Garapin, F. et al (1981) J. Mol. Biol. 150:1-14) and als
or pat, which confer resistance to chlorsulfuron and
phosphinotricin acetyltransferase, respectively (Murry, supra).
Additional selectable genes have been described, for example, trpB,
which allows cells to utilise indole in place of tryptophan, or
hisD, which allows cells to utilise histinol in place of histidine
(Hartman, S. C. and R. C. Mulligan (1988) Proc. Natl. Acad. Sci.
85:8047-51). Recently, the use of visible markers has gained
popularity with such markers as anthocyanins, .beta.-glucuronidase
and its substrate GUS, and luciferase and its substrate luciferin,
being widely used not only to identify transformants, but also to
quantify the amount of transient or stable protein expression
attributable to a specific vector system (Rhodes, C. A. et al.
(1995) Methods Mol. Biol. 55:121-131).
[0056] In vivo, the enzymatic kinase activity of the unmodified
polypeptides of Mnk towards a substrate can be enhanced by
appropriate stimuli, triggering the phosphorylation of Mnk. This
may be induced in the natural context by extracellular or
intracellular stimuli, such as signaling molecules or environmental
influences. One may generate a system containing actived Mnk, may
it be an organism, a tissue, a culture of cells or cell-free
environment, by exogenously applying this stimulus or by mimicking
this stimulus by a variety of the techniques, some of them
described further below. A system containing activated Mnk may be
produced (i) for the purpose of diagnosis, study, prevention, and
treatment of diseases and disorders related to body-weight
regulation and thermogenesis, for example, but not limited to,
metabolic diseases such as obesity, as well as related disorders
such as eating disorder, cachexia, diabetes mellitus, hypertension,
coronary heart disease, hypercholesterolemia, dyslipidemia,
osteoarthritis, gallstones, and sleep apnea, and disorders related
to ROS defence, such as diabetes mellitus, neurodegenerative
disorders, and cancer, e.g. cancers of the reproductive organs,
(ii) for the purpose of identifying or validating therapeutic
candidate agents, pharmaceuticals or drugs that influence the genes
of the invention or their encoded polypeptides, (iii) for the
purpose of generating cell lysates containing activated
polypeptides encoded by the genes of the invention, (iv) for the
purpose of isolating from this source activated polypeptides
encoded by the genes of the invention.
[0057] In one embodiment of the invention, one may produce
activated Mnk independent of the natural stimuli for the above said
purposes by, for example, but not limited to, (i) an agent that
mimics the natural stimulus; (ii) an agents, that acts downstream
of the natural stimulus, such as activators of the MAP kinase
pathway, phorbol ester, anisomycin, constitutive active allels of
the MAP kinase kinase kinases, of the MAP kinase kinases, of the
MAP kinase or Mnk itself as they are described or may be developed;
(iii) by introduction of single or multiple amino acid
substitutions, deletions or insertions within the sequence of Mnk
to yield constitutive active forms; (iv) by the use of isolated
fragments of Mnk. In addition, one may generate enzymatically
active Mnk in an ectopic system, prokaryotic or eukaryotic, in vivo
or in vitro, by co-transfering the activating components to this
system. These could be, for example, but not limited to, components
of the MAP kinase pathway such as constitutive active alleles of
the MAP kinase kinases Mek1 or Mkk6, together with the MAP kinases
ERK1 or ERK2 or the p38 MAPK isoforms. For example, one may
activate isolated Mnk protein in solution with a mutant polypeptide
of Mek1 containing the amino acid substitutions S218D and S222E
together with isolated ERK2 kinase in the presence of 1.0 mM
adenosine triphosphate and suitable buffer conditions such as 50 mM
N-(2-Hydroxyethyl)-piperazine-N'-(2-ethanesuflonic acid)/potassium
hydroxide pH 7.4, 5 mM magnesium chloride, 0.5 mM dithiothreitol
(see FIG. 14).
[0058] Although the presence/absence of marker gene expression
suggests that the gene of interest is also present, its presence
and expression may need to be confirmed. For example, if the
sequences encoding Mnk are inserted within a marker gene sequence,
recombinant cells containing sequences encoding Mnk can be
identified by the absence of marker gene function. Alternatively, a
marker gene can be placed in tandem with sequences encoding Mnk
under the control of a single promoter. Expression of the marker
gene in response to induction or selection usually indicates
expression of the tandem gene as well. Alternatively, host cells,
which contain the nucleic acid sequences encoding Mnk and express
Mnk, may be identified by a variety of procedures known to those of
skill in the art. These procedures include, but are not limited to,
DNA-DNA, or DNA-RNA hybridisation and protein bioassay or
immunoassay techniques, which include membrane, solution, or chip
based technologies for the detection and/or quantification of
nucleic acid or protein.
[0059] The presence of polynucleotide sequences encoding Mnk can be
detected by DNA-DNA or DNA-RNA hybridisation or amplification using
probes or portions or fragments of polynucleotides encoding Mnk.
Nucleic acid amplification based assays involve the use of
oligonucleotides or oligomers based on the sequences encoding Mnk
to detect transformants containing DNA or RNA encoding Mnk. As used
herein "oligonucleotides" or "oligomers" refer to a nucleic acid
sequence of at least about 10 nucleotides and as many as about 60
nucleotides, preferably about 15 to 30 nucleotides, and more
preferably about 20-25 nucleotides, which can be used as a probe or
amplimer.
[0060] A variety of protocols for detecting and measuring the
expression of Mnk, using either polyclonal or monoclonal antibodies
specific for the protein are known in the art. Examples include
enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA),
and fluorescence activated cell sorting (FACS). A two-site,
monoclonal-based immunoassay utilising monoclonal antibodies
reactive to two non-interfering epitopes on Mnk is preferred, but a
competitive binding assay may be employed. These and other assays
are described, among other places, in Hampton, R. et al. (1990;
Serological Methods, a Laboratory Manual, APS Press, St Paul,
Minn.) and Maddox, D. E. et al. (1983; J. Exp. Med.
158:1211-1216).
[0061] A wide variety of labels and conjugation techniques are
known by those skilled in the art and may be used in various
nucleic acid and amino acid assays. Means for producing labelled
hybridisation or PCR probes for detecting sequences related to
polynucleotides encoding Mnk include oligo-labelling, nick
translation, end-labelling or PCR amplification using a labelled
nucleotide.
[0062]
[0063] Alternatively, the sequences encoding Mnk, or any portions
thereof may be cloned into a vector for the production of an mRNA
probe. Such vectors are known in the art, are commercially
available, and may be used to synthesise RNA probes in vitro by
addition of an appropriate RNA polymerase such as T7, T3, or SP6
and labelled nucleotides. These procedures may be conducted using a
variety of commercially available kits (Pharmacia & Upjohn,
(Kalamazoo, Mich.); Promega (Madison Wis.); and U.S. Biochemical
Corp., (Cleveland, Ohio).
[0064] Suitable reporter molecules or labels, which may be used,
include radionuclides, enzymes, fluorescent, chemiluminescent, or
chromogenic agents as well as substrates, co-factors, inhibitors,
magnetic particles, and the like.
[0065] Host cells transformed with nucleotide sequences encoding
Mnk may be cultured under conditions suitable for the expression
and recovery of the protein from cell culture. The protein produced
by a recombinant cell may be secreted or contained intracellularly
depending on the sequence and/or the vector used. As will be
understood by those of skill in the art, expression vectors
containing polynucleotides which encode Mnk may be designed to
contain signal sequences, which direct secretion of Mnk through a
prokaryotic or eukaryotic cell membrane. Other recombinant
constructions may be used to join sequences encoding Mnk to
nucleotide sequence encoding a polypeptide domain, which will
facilitate purification of soluble proteins. Such purification
facilitating domains include, but are not limited to, metal
chelating peptides such as histidine-tryptophan modules that allow
purification on immobilised metals, protein A domains that allow
purification on immobilised immunoglobulin, and the domain utilised
in the FLAG extension/affinity purification system (Immunex Corp.,
Seattle, Wash.) The inclusion of cleavable linker sequences such as
those specific for Factor XA or Enterokinase (Invitrogen, San
Diego, Calif.) between the purification domain and Mnk may be used
to facilitate purification. One such expression vector provides for
expression of a fusion protein containing Mnk and a nucleic acid
encoding 6 histidine residues preceding a Thioredoxine or an
Enterokinase cleavage site. The histidine residues facilitate
purification on IMIAC (immobilised metal ion affinity
chromatography as described in Porath, J. et al. (1992, Prot. Exp.
Purif. 3: 263-281)) while the Enterokinase cleavage site provides a
means for purifying Mnk from the fusion protein. A discussion of
vectors which contain fusion proteins is provided in Kroll, D. J.
et al. (1993; DNA Cell Biol. 12:441-453). In addition to
recombinant production, fragments of Mnk may be produced by direct
peptide synthesis using solid-phase techniques (Merrifield J.
(1963) J. Am. Chem. Soc. 85:2149-2154). Protein synthesis may be
performed using manual techniques or by automation. Automated
synthesis may be achieved, for example, using Applied Biosystems
431A peptide synthesiser (Perkin Elmer). Various fragments of Mnk
may be chemically synthesised separately and combined using
chemical methods to produce the full length molecule.
[0066] Diagnostics and Therapeutics
[0067] The data disclosed in this invention show that the nucleic
acids and proteins of the invention and effector molecules thereof
are useful in diagnostic and therapeutic applications implicated,
for example but not limited to, in metabolic disorders like
obesity, diabetes, eating disorders, wasting syndromes (cachexia),
pancreatic dysfunctions, arteriosclerosis, coronary artery disease
(CAD), and other diseases and disorders as described above. Hence,
diagnostic and therapeutic uses for the Mnk proteins of the
invention are, for example but not limited to, the following: (i)
protein therapeutic, (ii) small molecule drug target, (iii)
antibody target (therapeutic, diagnostic, drug targeting/cytotoxic
antibody), (iv) diagnostic and/or prognostic marker, (v) gene
therapy (gene delivery/gene ablation), (vi) research tools, and
(vii) tissue regeneration in vitro and in vivo (regeneration for
all these tissues and cell types composing these tissues and cell
types derived from these tissues).
[0068] The nucleic acids and proteins of the invention are useful
in diagnostic and therapeutic applications implicated in various
diseases and disorders described above and/or other pathologies and
disorders. For example, but not limited to, cDNAs encoding the Mnk
proteins of the invention and particularly their human homologues
may be useful in gene therapy, and the Mnk proteins of the
invention and particularly their human homologues may be useful
when administered to a subject in need thereof. By way of
non-limiting example, the compositions of the present invention
will have efficacy for treatment of patients suffering from, for
example, but not limited to, in metabolic disorders like obesity,
diabetes, eating disorders, wasting syndromes (cachexia),
pancreatic dysfunctions, arteriosclerosis, coronary artery disease
(CAD), and other diseases and disorders, particularly as described
above.
[0069] The nucleic acid(s) encoding the Mnk protein(s) of the
invention, or fragments thereof, may further be useful in
diagnostic applications, wherein the presence or amount of the
nucleic acids or the proteins are to be assessed. These materials
are further useful in the generation of antibodies that bind
immunospecifically to the novel substances of the invention for use
in therapeutic or diagnostic methods.
[0070] For example, in one aspect, antibodies which are specific
for Mnk may be used directly as an antagonist, or indirectly as a
targeting or delivery mechanism for bringing a pharmaceutical agent
to cells or tissue which express Mnk. The antibodies may be
generated using methods that are well known in the art. Such
antibodies may include, but are not limited to, polyclonal,
monoclonal, chimerical, single chain, Fab fragments, and fragments
produced by a Fab expression library. Neutralising antibodies,
(i.e., those which inhibit dimer formation) are especially
preferred for therapeutic use.
[0071] For the production of antibodies, various hosts including
goats, rabbits, rats, mice, humans, and others, may be immunised by
injection with Mnk any fragment or oligopeptide thereof which has
immunogenic properties. Depending on the host species, various
adjuvants may be used to increase immunological response. Such
adjuvants include, but are not limited to, Freund's, mineral gels
such as aluminium hydroxide, and surface active substances such as
lysolecithin, pluronic polyols, polyanions, peptides, oil
emulsions, keyhole limpet hemocyanin, and dinitrophenol. Among
adjuvants used in human, BCG (Bacille Calmette-Guerin) and
Corynebacterium parvum are especially preferable. It is preferred
that the peptides, fragments, or oligopeptides used to induce
antibodies to Mnk have an amino acid sequence consisting of at
least five amino acids, and more preferably at least 10 amino
acids. It is preferable that they are identical to a portion of the
amino acid sequence of the natural protein, and they may contain
the entire amino acid sequence of a small, naturally occurring
molecule. Short stretches of Mnk amino acids may be fused with
those of another protein such as keyhole limpet hemocyanin and
antibody produced against the chimeric molecule.
[0072] Monoclonal antibodies to Mnk may be prepared using any
technique which provides for the production of antibody molecules
by continuous cell lines in culture. These include, but are not
limited to, the hybridoma technique, the human B-cell hybridoma
technique, and the EBV-hybridoma technique (Kohler, G. et al.
(1975) Nature 256:495-497; Kozbor, D., et al. (1985) J. Immunol.
Methods 81:31-42; Cote, R. J. et al. Proc. Natl. Acad. Sci.
80:2026-2030; Cole, S. P. et al. (1984) Mol. Cell Biol.
62:109-120).
[0073] In addition, techniques developed for the production of
"chimeric antibodies", the splicing of mouse antibody genes to
human antibody genes to obtain a molecule with appropriate antigen
specificity and biological activity can be used (Morrison, S. L. et
al. (1984) Proc. Natl. Acad. Sci. 81:6851-6855; Neuberger, M. S. et
al (1984) Nature 312:604-608; Takeda, S. et al. (1985) Nature
314:452-454). Alternatively, techniques described for the
production of single chain antibodies may be adapted, using methods
known in the art, to produce Mnk-specific single chain antibodies.
Antibodies with related specificity, but of distinct idiotypic
composition, may be generated by chain shuffling from random
combinatorial immunoglobulin libraries (Burton, D. R. (1991) Proc.
Natl. Acad. Sci. 88:11120-3). Antibodies may also be produced by
inducing in vivo production in the lymphocyte population or by
screening recombinant immunoglobulin libraries or panels of highly
specific binding reagents as disclosed in the literature (Orlandi,
R. et al. (1989) Proc. Natl. Acad. Sci. 86:3833-3837; Winter, G. et
al. (1991) Nature 349:293-299).
[0074] Antibody fragments, which contain specific binding sites for
Mnk, may also be generated. For example, such fragments include,
but are not limited to proteolytic fragments, e.g. the F(ab').sub.2
fragments which can be produced by Pepsin digestion of the antibody
molecule and the Fab fragments which can be generated by reducing
the disulfide bridges of F(ab').sub.2 fragments. Alternatively,
recombinant fragments may be generated. For example, Fab expression
libraries may be constructed to allow rapid and easy identification
of monoclonal Fab fragments with the desired specificity (Huse, W.
D. et al. (1989) Science 254:1275-1281).
[0075] Various immunoassays may be used for screening to identify
antibodies having the desired specificity. Numerous protocols for
competitive binding and immunoradiometric assays using either
polyclonal or monoclonal antibodies with established specificities
are well known in the art. Such immunoassays typically involve the
measurement of complex formation between Mnk and its specific
antibody. A two-site, monoclonal-based immunoassay utilising
monoclonal antibodies reactive to two non-interfering Mnk epitopes
is preferred, but a competitive binding assay may also be employed
(Maddox, supra).
[0076] In another embodiment of the invention, the Mnk
polynucleotides or any fragment thereof, or nucleic acid effector
molecules, aptamers, anti-sense molecules, ribozymes or RNAi
molecules, may be used for therapeutic purposes. In one aspect,
aptamers, i.e. nucleic acid molecules, which are capable of binding
to a Mnk protein and modulating its activity, may be generated by a
screening and selection procedure involving the use of
combinational nucleic acid libraries.
[0077] In a further aspect, antisense molecules to the
polynucleotide encoding Mnk may be used in situations in which it
would be desirable to block the transcription of the mRNA. In
particular, cells may be transformed with sequences complementary
to polynucleotides encoding Mnk. Thus, antisense molecules may be
used to modulate Mnk activity, or to achieve regulation of gene
function. Such technology is now well know in the art, and sense or
antisense oligomers or larger fragments, can be designed from
various locations along the coding or control regions of sequences
encoding Mnk. Expression vectors derived from retroviruses,
adenovirus, herpes or vaccinia viruses, or from various bacterial
plasmids may be used for delivery of nucleotide sequences to the
targeted organ, tissue or cell population. Methods, which are well
known to those skilled in the art, can be used to construct
recombinant vectors, which will express antisense molecules
complementary to the polynucleotides of the gene encoding Mnk.
These techniques are described both in Sambrook et al. (supra) and
in Ausubel et al. (supra). Genes encoding Mnk can be turned off by
transforming a cell or tissue with expression vectors which express
high levels of polynucleotide or fragment thereof which encodes
Mnk. Such constructs may be used to introduce untranslatable sense
or antisense sequences into a cell. Even in the absence of
integration into the DNA, such vectors may continue to transcribe
RNA molecules until they are disabled by endogenous nucleases.
Transient expression may last for a month or more with a
non-replicating vector and even longer if appropriate replication
elements are part of the vector system.
[0078] As mentioned above, modifications of gene expression can be
obtained by designing antisense molecules, e.g. DNA, RNA, or
nucleic acid analogues such as PNA, to the control regions of the
gene encoding Mnk, i.e., the promoters, enhancers, and introns.
Oligonucleotides derived from the transcription initiation site,
e.g., between positions -10 and +10 from the start site, are
preferred. Similarly, inhibition can be achieved using "triple
helix" base-pairing methodology. Triple helix pairing is useful
because it cause inhibition of the ability of the double helix to
open sufficiently for the binding of polymerases, transcription
factors, or regulatory molecules. Recent therapeutic advances using
triplex DNA have been described in the literature (Gee, J. E. et
al. (1994) In; Huber, B. E. and B. I. Carr, Molecular and
Immunologic Approaches, Futura Publishing Co., Mt. Kisco, N.Y.).
The antisense molecules may also be designed to block translation
of mRNA by preventing the transcript from binding to ribosomes.
[0079] Ribozymes, enzymatic RNA molecules, may also be used to
catalyse the specific cleavage of RNA. The mechanism of ribozyme
action involves sequence-specific hybridisation of the ribozyme
molecule to complementary target RNA, followed by endonucleolytic
cleavage. Examples, which may be used, include engineered
hammerhead motif ribozyme molecules that can be specifically and
efficiently catalyse endonucleolytic cleavage of sequences encoding
Mnk. Specific ribozyme cleavage sites within any potential RNA
target are initially identified by scanning the target molecule for
ribozyme cleavage sites which include the following sequences: GUA,
GUU, and GUC. Once identified, short RNA sequences of between 15
and 20 ribonucleotides corresponding to the region of the target
gene containing the cleavage site may be evaluated for secondary
structural features which may render the oligonucleotide
inoperable. The suitability of candidate targets may also be
evaluated by testing accessibility to hybridisation with
complementary oligonucleotides using ribonuclease protection
assays.
[0080] Effector molecules, e.g. antisense molecules and ribozymes
of the invention may be prepared by any method known in the art for
the synthesis of nucleic acid molecules. These include techniques
for chemically synthesising oligonucleotides such as solid phase
phosphoramidite chemical synthesis. Alternatively, RNA molecules
may be generated by in vitro and in vivo transcription of DNA
sequences encoding Mnk. Such DNA sequences may be incorporated into
a variety of vectors with suitable RNA polymerase promoters such as
T7 or SP6. Alternatively, these cDNA constructs that synthesise
antisense RNA constitutively or inducibly can be introduced into
cell lines, cells, or tissues. RNA molecules may be modified to
increase intracellular stability and half-life. Possible
modifications include, but are not limited to, the addition of
flanking sequences at the 5' and/or 3' ends of the molecule or the
use of phosphorothioate or 2' O-methyl rather than
phosphodiesterase linkages within the backbone of the molecule.
This concept is inherent in the production of PNAs and can be
extended in all of these molecules by the inclusion of
non-traditional bases such as inosine, queosine, and wybutosine, as
well as acetyl-, methyl-, thio-, and similarly modified forms of
adenine, cytidine, guanine, thymine, and uridine which are not as
easily recognised by endogenous endonucleases;
[0081] The activity of Mnk proteins can be assayed for example by
in vitro kinase assays, as described by Tschopp et al., 2000, supra
or any other suitable assay principle as described below. As
inhibitor of Mnk in this assay, a staurosporine derivative such as
CGP57380 or CGP052088 can be used, as described by Tschopp et al.,
2000, supra or Knauf et al., 2001, supra. As negative control, the
compound CGP52428 which is inactive against Mnk, but displays a
similar cytotoxicity as CGP052088, or any other chemical entities
with kinase inhibitory activity with exception of activity against
Mnk may be used. Moreover, derivatives of CGP57380 can be assayed
for activity against Mnk and are substances for the treatment,
prophylaxis, and diagnosis of metabolic diseases as mentioned
above. Derivatives of CGP57380 could for example be generated by
modification through conventional chemical, physical and
biochemical means, and may be used to produce combinatorial
libraries. They may be subjected to directed or random chemical
modifications, such as acylation, alkylation, esterification,
amidification, etc. to produce structural analogs.
[0082] Further, the invention relates to the use of Mnk kinase
inhibitors or activators for the treatment, prophylaxis or
diagnosis of metabolic diseases as mentioned above. Preferably, but
not exclusively, the Mnk kinase inhibitors are staurosporine or
pyrazole derivatives. Examples of pyrazole derivatives are
described in EP-A-0 819 129 which is herein incorporated by
reference. Since CGP57380 is not cytotoxic up to 30 .mu.M, this
substance may be preferably used to inhibit kinase activity,
preferably Mnk2, and used as substance for the treatment,
prophylaxis, and diagnosis of metabolic diseases as mentioned
above.
[0083] Many methods for introducing vectors into cells or tissues
are available and equally suitable for use in vivo, in vitro, and
ex vivo. For ex vivo therapy, vectors may be introduced into stem
cells taken from the patient and clonally propagated for autologous
transplant back into that same patient. Delivery by transfection
and by liposome injections may be achieved using methods, which are
well known in the art. Any of the therapeutic methods described
above may be applied to any suitable subject including, for
example, mammals such as dogs, cats, cows, horses, rabbits,
monkeys, and most preferably, humans.
[0084] An additional embodiment of the invention relates to the
administration of a pharmaceutical composition, in conjunction with
a pharmaceutically acceptable carrier, for any of the therapeutic
effects discussed above. Such pharmaceutical compositions may
consist of Mnk, antibodies to Mnk, mimetics, agonists, antagonists,
or inhibitors of Mnk. The compositions may be administered alone or
in combination with at least one other agent, such as stabilising
compound, which may be administered in any sterile, biocompatible
pharmaceutical carrier, including, but not limited to, saline,
buffered saline, dextrose, and water. The compositions may be
administered to a patient alone, or in combination with other
agents, drugs or hormones. The pharmaceutical compositions utilised
in this invention may be administered by any number of routes
including, but not limited to, oral, intravenous, intramuscular,
intra-arterial, intramedullary, intrathecal, intraventricular,
transdermal, subcutaneous, intraperitoneal, intranasal, enteral,
topical, sublingual, or rectal means.
[0085] In addition to the active ingredients, these pharmaceutical
compositions may contain suitable pharmaceutically-acceptable
carriers comprising excipients and auxiliaries, which facilitate
processing of the active compounds into preparations which, can be
used pharmaceutically. Further details on techniques for
formulation and administration may be found in the latest edition
of Remington's Pharmaceutical Sciences (Maack Publishing Co.,
Easton, Pa.). Pharmaceutical compositions for oral administration
can be formulated using pharmaceutically acceptable carriers well
known in the art in dosages suitable for oral administration. Such
carriers enable the pharmaceutical compositions to be formulated as
tablets, pills, dragees, capsules, liquids, gels, syrups, slurries,
suspensions, and the like, for ingestion by the patient.
[0086] The pharmaceutical compositions of the present invention may
be manufactured in a manner that is known in the art, e.g., by
means of conventional mixing, dissolving, granulating,
dragee-making, levigating, emulsifying, encapsulating, entrapping,
or lyophilising processes. The pharmaceutical composition may be
provided as a salt and can be formed with many acids, including but
not limited to, hydrochloric, sulphuric, acetic, lactic, tartaric,
malic, succinic, etc. After pharmaceutical compositions have been
prepared, they can be placed in an appropriate container and
labelled for treatment of an indicated condition. For
administration of Mnk, such labelling would include amount,
frequency, and method of administration.
[0087] Pharmaceutical compositions suitable for use in the
invention include compositions wherein the active ingredients are
contained in an effective amount to achieve the intended purpose.
The determination of an effective dose is well within the
capability of those skilled in the art. For any compounds, the
therapeutically effective does can be estimated initially either in
cell culture assays, e.g., of preadipocyte cell lines, or in animal
models, usually mice, rabbits, dogs, or pigs. The animal model may
also be used to determine the appropriate concentration range and
route of administration. Such information can then be used to
determine useful doses and routes for administration in humans. A
therapeutically effective dose refers to that amount of active
ingredient, for example Mnk fragments thereof, antibodies of Mnk,
to treat a specific condition. Therapeutic efficacy and toxicity
may be determined by standard pharmaceutical procedures in cell
cultures or experimental animals, e.g., ED50 (the dose
therapeutically effective in 50% of the population) and LD50 (the
dose lethal to 50% of the population). The dose ratio between
therapeutic and toxic effects is the therapeutic index, and it can
be expressed as the ratio, LD50/ED50. Pharmaceutical compositions,
which exhibit large therapeutic indices, are preferred. The data
obtained from cell culture assays and animal studies is used in
formulating a range of dosage for human use. The dosage contained
in such compositions is preferably within a range of circulating
concentrations that include the ED50 with little or no toxicity.
The dosage varies within this range depending upon the dosage from
employed, sensitivity of the patient, and the route of
administration. The exact dosage will be determined by the
practitioner, in light of factors related to the subject that
requires treatment. Dosage and administration are adjusted to
provide sufficient levels of the active moiety or to maintain the
desired effect. Factors, which may be taken into account, include
the severity of the disease state, general health of the subject,
age, weight, and gender of the subject, diet, time and frequency of
administration, drug combination(s), reaction sensitivities, and
tolerance/response to therapy. Long-acting pharmaceutical
compositions may be administered every 3 to 4 days, every week, or
once every two weeks depending on half-life and clearance rate of
the particular formulation. Normal dosage amounts may vary from 0.1
to 100,000 micrograms, up to a total dose of about 1 g, depending
upon the route of administration. Guidance as to particular dosages
and methods of delivery is provided in the literature and generally
available to practitioners in the art. Those skilled in the art
employ different formulations for nucleotides than for proteins or
their inhibitors. Similarly, delivery of polynucleotides or
polypeptides will be specific to particular cells, conditions,
locations, etc.
[0088] In another embodiment, antibodies which specifically bind
Mnk may be used for the diagnosis of conditions or diseases
characterised by or associated with over- or underexpression of
Mnk, or in assays to monitor patients being treated with Mnk,
agonists, antagonists or inhibitors. The antibodies useful for
diagnostic purposes may be prepared in the same manner as those
described above for therapeutics. Diagnostic assays for Mnk include
methods, which utilise the antibody and a label to detect Mnk in
human body fluids or extracts of cells or tissues. The antibodies
may be used with or without modification, and may be labelled by
joining them, either covalently or non-covalently, with a reporter
molecule. A wide variety of reporter molecules, which are known in
the art may be used several of which are described above.
[0089] A variety of protocols including ELISA, RIA, and FACS for
measuring Mnk are known in the art and provide a basis for
diagnosing altered or abnormal levels of Mnk expression. Normal or
standard values for Mnk expression are established by combining
body fluids or cell extracts taken from normal mammalian subjects,
preferably human, with antibody to Mnk under conditions suitable
for complex formation. The amount of standard complex formation may
be quantified by various methods, but preferably by photometric,
means. Quantities of Mnk expressed in control and disease samples
e.g. from biopsied tissues are compared with the standard values.
Deviation between standard and subject values establishes the
parameters for diagnosing disease. Analysis of Mnk expression can
also be performed by determination of Mnk activity in assay formats
well known in the art and described in more detail below.
[0090] In another embodiment of the invention, the polynucleotides
specific for Mnk may be used for diagnostic purposes. The
polynucleotides, which may be used, include oligonucleotide
sequences, antisense RNA and DNA molecules, and PNAs. The
polynucleotides may be used to detect and quantitate gene
expression in biopsied tissues in which expression of Mnk may be
correlated with disease. The diagnostic assay may be used to
distinguish between absence, presence, and excess expression of
Mnk, and to monitor regulation of Mnk levels during therapeutic
intervention.
[0091] In one aspect, hybridisation with PCR probes which are
capable of detecting polynucleotide sequences, including genomic
sequences, encoding Mnk and/or closely related molecules, may be
used to identify nucleic acid sequences which encode Mnk. The
specificity of the probe, whether it is made from a highly specific
region, e.g., unique nucleotides in the 5' regulatory region, or a
less specific region, e.g., especially in the 3' coding region, and
the stringency of the hybridisation or amplification (maximal,
high, intermediate, or low) will determine whether the probe
identifies only naturally occurring sequences encoding Mnk,
alleles, or related sequences. Probes may also be used for the
detection of related sequences, and should preferably contain at
least 50% of the nucleotides from any of the Mnk encoding
sequences. The hybridisation probes of the subject invention may be
DNA or RNA and derived from the nucleotide sequence of AF237775,
NM.sub.--017572.1, NM.sub.--003684.2, or AB000409.1 or from a
genomic sequence including promoter, enhancer elements, and introns
of the naturally occurring Mnk. Means for producing specific
hybridisation probes for DNAs encoding Mnk include the cloning of
nucleic acid sequences encoding Mnk derivatives into vectors for
the production of mRNA probes. Such vectors are known in the art,
commercially available, and may be used to synthesise RNA probes in
vitro by means of the addition of the appropriate RNA polymerases
and the appropriate labelled nucleotides. Hybridisation probes may
be labelled by a variety of reporter groups, for example,
radionuclides such as .sup.32P or .sup.35S, or enzymatic labels,
such as alkaline phosphatase coupled to the probe via avidin/biotin
coupling systems, and the like.
[0092] Polynucleotide sequences encoding Mnk may be used for the
diagnosis of conditions or diseases, which are associated with
expression of Mnk. Examples of such conditions or diseases include,
but are not limited to, pancreatic diseases and disorders,
including diabetes. Polynucleotide sequences encoding Mnk may also
be used to monitor the progress of patients receiving treatment for
pancreatic diseases and disorders, including diabetes. The
polynucleotide sequences encoding Mnk may be used in Southern or
Northern analysis, dot blot, or other membrane-based technologies;
in PCR technologies; or in dip stick, pin, ELISA or chip assays
utilising fluids or tissues from patient biopsies to detect altered
Mnk expression. Such qualitative or quantitative methods are well
known in the art.
[0093] In a particular aspect, the nucleotide sequences encoding
Mnk may be useful in assays that detect activation or induction of
various metabolic diseases and disorders, including obesity,
diabetes, eating disorders, wasting syndromes (cachexia),
pancreatic dysfunctions, arteriosclerosis, coronary artery disease
(CAD), disorders related to ROS production, and neurodegenerative
diseases. The nucleotide sequences encoding Mnk may be labelled by
standard methods, and added to a fluid or tissue sample from a
patient under conditions suitable for the formation of
hybridisation complexes. After a suitable incubation period, the
sample is washed and the signal is quantitated and compared with a
standard value. The presence of altered levels of nucleotide
sequences encoding Mnk in the sample compared to a control sample
indicates the presence of the associated disease. Such assays may
also be used to evaluate the efficacy of a particular therapeutic
treatment regimen in animal studies, in clinical trials, or in
monitoring the treatment of an individual patient.
[0094] In order to provide a basis for the diagnosis of disease
associated with expression of Mnk, a normal or standard profile for
expression is established. This may be accomplished by combining
body fluids or cell extracts taken from normal subjects, either
animal or human, with a sequence, or a fragment thereof, which
encodes Mnk, under conditions suitable for hybridisation or
amplification. Standard hybridisation may be quantified by
comparing the values obtained from normal subjects with those from
an experiment where a known amount of a substantially purified
polynucleotide is used. Standard values obtained from normal
samples may be compared with values obtained from samples from
patients who are symptomatic for disease. Deviation between
standard and subject values is used to establish the presence of
disease. Once disease is established and a treatment protocol is
initiated, hybridisation assays may be repeated on a regular basis
to evaluate whether the level of expression in the patient begins
to approximate that, which is observed in the normal patient. The
results obtained from successive assays may be used to show the
efficacy of treatment over a period ranging from several days to
months.
[0095] With respect to metabolic diseases and disorders, including
obesity, diabetes, eating disorders, wasting syndromes (cachexia),
pancreatic dysfunctions, arteriosclerosis, coronary artery disease
(CAD), disorders related to ROS production, and neurodegenerative
diseases presence of a relatively high amount of transcript in
biopsied tissue from an individual may indicate a predisposition
for the development of the disease, or may provide a means for
detecting the disease prior to the appearance of actual clinical
symptoms. A more definitive diagnosis of this type may allow health
professionals to employ preventative measures or aggressive
treatment earlier thereby preventing the development or further
progression of the pancreatic diseases and disorders. Additional
diagnostic uses for oligonucleotides designed from the sequences
encoding Mnk may involve the use of PCR. Such oligomers may be
chemically synthesised, generated enzymatically, or produced from a
recombinant source. Oligomers will preferably consist of two
nucleotide sequences, one with sense orientation (5'.fwdarw.3') and
another with antisense (3'.rarw.5'), employed under optimised
conditions for identification of a specific gene or condition. The
same two oligomers, nested sets of oligomers, or even a degenerate
pool of oligomers may be employed under less stringent conditions
for detection and/or quantification of closely related DNA or RNA
sequences.
[0096] Methods which may also be used to quantitate the expression
of Mnk include radiolabelling or biotinylating nucleotides,
coamplification of a control nucleic acid, and standard curves onto
which the experimental results are interpolated (Melby, P. C. et
al. (1993) J. Immunol. Methods, 159:235-244; Duplaa, C. et al.
(1993) Anal. Biochem. 212:229-236). The speed of quantification of
multiple samples may be accelerated by running the assay in an
ELISA format where the oligomer of interest is presented in various
dilutions and a spectrophotometric or colorimetric response gives
rapid quantification.
[0097] In another embodiment of the invention, the nucleic acid Mnk
sequences may also be used to generate hybridisation probes, which
are useful for mapping the naturally occurring genomic sequence.
The sequences may be mapped to a particular chromosome or to a
specific region of the chromosome using well known techniques. Such
techniques include FISH, FACS, or artificial chromosome
constructions, such as yeast artificial chromosomes, bacterial
artificial chromosomes, bacterial P1 constructions or single
chromosome cDNA libraries as reviewed in Price, C. M. (1993) Blood
Rev. 7:127-134, and Trask, B. J. (1991) Trends Genet. 7:149-154.
FISH (as described in Verma et al. (1988) Human Chromosomes: A
Manual of Basic Techniques, Pergamon Press, New York, N.Y.) may be
correlated with other physical chromosome mapping techniques and
genetic map data. Examples of genetic map data can be found in the
1994 Genome Issue of Science (265:1981 f). Correlation between the
location of the gene encoding Mnk on a physical chromosomal map and
a specific disease, or predisposition to a specific disease, may
help to delimit the region of DNA associated with that genetic
disease.
[0098] The nucleotide sequences of the subject invention may be
used to detect differences in gene sequences between normal,
carrier or affected individuals. In situ hybridization of
chromosomal preparations and physical mapping techniques such as
linkage analysis using established chromosomal markers may be used
for extending genetic maps. Often the placement of a gene on the
chromosome of another mammalian species, such as mouse, may reveal
associated markers even if the number or arm of a particular human
chromosome is not known. New sequences can be assigned to
chromosomal arms or parts thereof, by physical mapping. This
provides valuable information to investigators searching for
disease genes using positional cloning or other gene discovery
techniques. Once the disease or syndrome has been crudely localized
by genetic linkage to a particular genomic region, for example, AT
to 11 q22-23 (Gatti, R. A. et al. (1988) Nature 336:577-580), any
sequences mapping to that area may represent associated or
regulatory genes for further investigation. The nucleotide
sequences of the subject invention may also be used to detect
differences in the chromosomal location due to translocation,
inversion, etc. among normal, carrier or affected individuals.
[0099] In another embodiment of the invention, the proteins of the
invention, its catalytic or immunogenic fragments or oligopeptides
thereof, an in vitro model, a genetically altered cell or animal,
can be used for screening libraries of compounds in any of a
variety of drug screening techniques. One can identify effectors,
e.g. receptors, enzymes, proteins, peptides, ligands or substrates
that bind to, modulate or mimic the action of one or more of the
proteins of the invention. The protein or fragment thereof employed
in such screening may be free in solution, affixed to a solid
support, borne on a cell surface, or located intracellularly. The
formation of binding complexes, between the proteins of the
invention and the agent tested, may be measured. Agents could also,
either directly or indirectly, influence the activity of the
proteins of the invention. Target mechanisms can for example
include a kinase activity, particularly the phosphorylation of
proteins or peptides, most preferably, but not limited to serine
and threonine residues. Another target mechanism could include the
regulation of Mnk function by posttranslational modifications such
as phosphorylation, dephosphorylation, acetylation, alkylation,
ubiquitination, proteolytic processing subcellular localization or
degradation. Yet another target mechanism could include the
interaction of Mnk with protein binding partners such as, but not
limited to, phospholipase A, estrogen receptors, kinases or
translation factors. Of particular interest are screening assays
for agents that have a low toxicity for mammalian cells. The term
"agent" as used herein describes any molecule, e.g. protein or
pharmaceutical, with the capability of altering or mimicking the
physiological function of one or more of the proteins of the
invention. Candidate agents encompass numerous chemical classes,
though typically they are organic molecules, preferably small
organic compounds having a molecular weight of more than 50 and
less than about 2,500 Daltons. Candidate agents comprise functional
groups necessary for structural interaction with proteins,
particularly hydrogen bonding, and typically include at least an
amine, carbonyl, hydroxyl or carboxyl group, preferably at least
two of the functional chemical groups. The candidate agents often
comprise carbocyclic or heterocyclic structures and/or aromatic or
polyaromatic structures substituted with one or more of the above
functional groups.
[0100] Candidate agents are also found among biomolecules including
peptides, saccharides, fatty acids, steroids, purines, pyrimidines,
nucleic acids and derivatives, structural analogs or combinations
thereof. Candidate agents are obtained from a wide variety of
sources including libraries of synthetic or natural compounds. For
example, numerous means are available for random and directed
synthesis of a wide variety of organic compounds and biomolecules,
including expression of randomized oligonucleotides and
oligopeptides. Alternatively, libraries of natural compounds in the
form of bacterial, fungal, plant and animal extracts are available
or readily produced. Additionally, natural or synthetically
produced libraries and compounds are readily modified through
conventional chemical, physical and biochemical means, and may be
used to produce combinatorial libraries. Known pharmacological
agents may be subjected to directed or random chemical
modifications, such as acylation, alkylation, esterification,
amidification, etc. to produce structural analogs. Where the
screening assay is a binding assay, one or more of the molecules
may be joined to a label, where the label can directly or
indirectly provide a detectable signal.
[0101] Another technique for drug screening, which may be used,
provides for high throughput screening of compounds having suitable
binding affinity to the protein of interest as described in
published PCT application WO84/03564. In this method, as applied to
the proteins of the invention large numbers of different small test
compounds, e.g. aptamers, peptides, low-molecular weight compounds
etc., are provided or synthesized on a solid substrate, such as
plastic pins or some other surface. The test compounds are reacted
with the proteins or fragments thereof, and washed. Bound proteins
are then detected by methods well known in the art. Purified
proteins can also be coated directly onto plates for use in the
aforementioned drug screening techniques. Alternatively,
non-neutralizing antibodies can be used to capture the peptide and
immobilize it on a solid support. In another embodiment, one
may-use competitive drug screening assays in which neutralizing
antibodies capable of binding the protein specifically compete with
a test compound for binding the protein. In this manner, the
antibodies can be used to detect the presence of any peptide, which
shares one or more antigenic determinants with the protein.
[0102] Candidate agents may also be found in kinase assays where a
kinase substrate such as a protein or a peptide, which may or may
not include modifications as further described below, or others are
phosphorylated by the proteins or protein fragments of the
invention. A therapeutic candidate agent may be identified by its
ability to increase or decrease the enzymatic activity of the
proteins of the invention. The kinase activity may be detected by
change of the chemical, physical or immunological properties of the
substrate due to phosphorylation.
[0103] One example could be the transfer of radioisotopically
labelled phosphate groups from an appropriate donor molecule to the
kinase substrate catalyzed by the polypeptides of the invention.
The phosphorylation of the substrate may be followed by detection
of the substrates autoradiography with techniques well known in the
art.
[0104] Yet in another example, the change of mass of the substrate
due to its phosphorylation may be detected by mass spectrometry
techniques.
[0105] One could also detect the phosphorylation status of a
substrate with a reagent discriminating between the phosphorylated
and unphosphorylated status of the substrate. Such a reagent may
act by having different affinities for the phosphorylated and
unphosphorylated forms of the substrate or by having specific
affinity for phosphate groups. Such a reagent could be, but is not
limited to, an antibody or antibody derivative, a recombinant
antibody-like structure, a protein, a nucleic acid, a molecule
containing a complexed metal ion, an anion exchange chromatography
matrix, an affinity chromatography matrix or any other molecule
with phosphorylation dependend selectivity towards the
substrate.
[0106] Such a reagent could be employed to detect the kinase
substrate, which is immobilized on a solid support during or after
an enzymatic reaction. If the reagent is an antibody, its binding
to the substrate could be detected by a variety of techniques as
they are described in Harlow and Lane, 1998, Antibodies, CSH Lab
Press, NY. If the reagent molecule is not an antibody, it may be
detected by virtue of its chemical, physical or immunological
properties, being endogenously associated with it or engineered to
it.
[0107] Yet in another example the kinase substrate may have
features, designed or endogenous, to facilitate its binding or
detection in order to generate a signal that is suitable for the
analysis of the substrates phosphorylation status. These features
may be, but are not limited to, a biotin molecule or derivative
thereof, a glutathione-S-transferase moiety, a moiety of six or
more consecutive histidine residues, an amino acid sequence or
hapten to function as an epitope tag, a fluorochrome, an enzyme or
enzyme fragment. The kinase substrate may be linked to these or
other features with a molecular spacer arm to avoid steric
hindrance.
[0108] In one example the kinase substrate may be labelled with a
fluorophore. The binding of the reagent to the labelled substrate
in solution may be followed by the technique of fluorescence
polarization as it is described in the literature (see, for
example, Deshpande, S. et al. (1999) Prog. Biomed. Optics (SPIE)
3603:261; Parker, G. J. et al. (2000) J. Biomol. Screen. 5:77-88;
Wu, P. et al. (1997) Anal. Biochem. 249:29-36). In a variation of
this example, a fluorescent tracer molecule may compete with the
substrate for the analyte to detect kinase activity by a technique
which is know to those skilled in the art as indirect fluorescence
polarization.
[0109] The nucleic acids encoding the proteins of the invention can
be used to generate transgenic cell lines and animals. These
transgenic animals are useful in the study of the function and
regulation of the proteins of the invention in vivo. Transgenic
animals, particularly mammalian transgenic animals, can serve as a
model system for the investigation of many developmental and
cellular processes common to humans. Transgenic animals may be made
through homologous recombination in embryonic stem cells, where the
normal locus of the gene encoding the protein of the invention is
mutated. Alternatively, a nucleic acid construct encoding the
protein is injected into oocytes and is randomly integrated into
the genome. One may also express the genes of the invention or
variants thereof in tissues where they are not normally expressed
or at abnormal times of development. Furthermore, variants of the
genes of the invention like specific constructs expressing
anti-sense molecules or expression of dominant negative mutations,
which will block or alter the expression of the proteins of the
invention may be randomly integrated into the genome. A detectable
marker, such as lac Z or luciferase may be introduced into the
locus of the genes of the invention, where upregulation of
expression of the genes of the invention will result in an easily
detectable change in phenotype. Vectors for stable integration
include plasmids, retroviruses and other animal viruses, yeast
artificial chromosomes (YACs), and the like. DNA constructs for
homologous recombination will contain at least portions of the
genes of the invention with the desired genetic modification, and
will include regions of homology to the target locus. Conveniently,
markers for positive and negative selection are included. DNA
constructs for random integration do not need to contain regions of
homology to mediate recombination. DNA constructs for random
integration will consist of the nucleic acids encoding the proteins
of the invention, a regulatory element (promoter), an intron and a
poly-adenylation signal. Methods for generating cells having
targeted gene modifications through homologous recombination are
known in the field. For embryonic stem (ES) cells, an ES cell line
may be employed, or embryonic cells may be obtained freshly from a
host, e.g. mouse, rat, guinea pig, etc. Such cells are grown on an
appropriate fibroblast-feeder layer and are grown in the presence
of leukemia inhibiting factor (LIF). ES or embryonic cells may be
transfected and can then be used to produce transgenic animals.
After transfection, the ES cells are plated onto a feeder layer in
an appropriate medium. Cells containing the construct may be
selected by employing a selection medium. After sufficient time for
colonies to grow, they are picked and analyzed for the occurrence
of homologous recombination. Colonies that are positive may then be
used for embryo manipulation and morula aggregation. Briefly,
morulae are obtained from 4 to 6 week old superovulated females,
the Zona Pellucida is removed and the morulae are put into small
depressions of a tissue culture dish. The ES cells are trypsinized,
and the modified cells are placed into the depression closely to
the morulae. On the following day the aggregates are transfered
into the uterine horns of pseudopregnant females. Females are then
allowed to go to term. Chimeric offsprings can be readily detected
by a change in coat color and are subsequently screened for the
transmission of the mutation into the next generation
(F1-generation). Offspring of the F1-generation are screened for
the presence of the modified gene and males and females having the
modification are mated to produce homozygous progeny. If the gene
alterations cause lethality at some point in development, tissues
or organs can be maintained as allogenic or congenic grafts or
transplants, or in vitro culture. The transgenic animals may be any
non-human mammal, such as laboratory animal, domestic animals,
etc., for example, mouse, rat, guinea pig, sheep, cow, pig, and
others. The transgenic animals may be used in functional studies,
drug screening, and other applications and are useful in the study
of the function and regulation of the proteins of the invention in
vivo.
[0110] Finally, the invention also relates to a kit comprising at
least one of
[0111] (a) a Mnk2 (Mnk2a or Mnk2b) or Mnk1 nucleic acid molecule or
a fragment thereof;
[0112] (b) a vector comprising the nucleic acid of (a);
[0113] (c) a host cell comprising the nucleic acid of (a) or the
vector of (b);.
[0114] (d) a polypeptide encoded by the nucleic acid of (a);
[0115] (e) a fusion polypeptide encoded by the nucleic acid of
(a);
[0116] (f) an antibody, an aptamer or another receptor against the
nucleic acid of (a) or the polypeptide of (d) or (e) and
[0117] (g) an anti-sense oligonucleotide of the nucleic acid of
(a).
[0118] The kit may be used for diagnostic or therapeutic purposes
or for screening applications as described above. The kit may
further contain user instructions.
THE FIGURES SHOW
[0119] FIG. 1 shows the increase of triglyceride content of
EP(3)3333 and EP(3)3576 male flies caused by homozygous viable
integration of the P-vector (in comparison to EP-control males).
Shown is the ratio of the triglyceride to protein content of the
mutants in percent (%)).
[0120] FIG. 2 shows the molecular organisation of the mutated LK6
gene locus. The dotted black line represents the position of the
cDNA (from position 7544500 to 7559500 on chromosome 3R) that
includes the integration sites of EP(3)3333 and EP(3)3576.
Transcribed DNA sequences (ESTs) and predicted exons are shown as
bars in the lower two lines. Predicted exons of gene CG17342
(GadFly, Lk6) are shown as dark grey bars and introns as light grey
bars. Lk6 encodes for a gene that is predicted by GadFly sequence
analysis programs as Gadfly Accession Number CG17342.
[0121] FIG. 3 shows the comparison of Mnk proteins.
[0122] FIG. 3A shows the comparison (CLUSTAL X 1.8) of Mnk proteins
from different species, hXP.sub.--030637 refers to human Mnk2
(identical to Genbank Accession No. AF237775), hXP.sub.--001600
refers to human Mnk1 (identical to Genbank Accession No.
AB000409.1), and AAB18789 refers to the protein encoded by
Drosophila Lk6 gene with GadFly Accession No. CG17342.
[0123] Gaps in the alignment are represented as --.
[0124] FIG. 3B shows the comparison (CLUSTAL W 1.82) of human Mnk2
proteins. Genbank Accession Number XM.sub.--030637.3 is identical
to Genbank Accession Number AF237775, and Genbank Accession Number
NM.sub.--017572.1 shows a different variant of the human Mnk2
protein.
[0125] FIG. 3C shows the comparison (CLUSTAL W 1.82) of human Mnk1
proteins. Genbank Accession Number XM.sub.--001600.2 is identical
to Genbank Accession Number AB000409.1, and Genbank Accession
Number NM.sub.--003684.2 shows a different variant of the Mnk1
protein.
[0126] FIG. 3D. Nucleic acid sequence of human MAP
kinase-interacting kinase (Mnk) 2a (SEQ ID NO: 1; GenBank Accession
Number AF237775, identical to GenBank Accession Number
XM.sub.--030637)
[0127] FIG. 3E. Amino Acid sequence of human MAP kinase-interacting
kinase (Mnk) 2a (SEQ ID NO: 2; GenBank Accession Number AF237775,
identical to GenBank Accession Number XM.sub.--030637)
[0128] FIG. 3F. Nucleic acid sequence of human MAP
kinase-interacting kinase (Mnk) 2b (SEQ ID NO: 3; GenBank Accession
Number AF237776 or NM.sub.--017572)
[0129] FIG. 3G. Amino Acid sequence of human MAP kinase-interacting
kinase (Mnk) 2b (SEQ ID NO: 4; GenBank Accession Number AF237776 or
NM.sub.--017572)
[0130] FIG. 3H. Nucleic acid sequence of human MAP
kinase-interacting kinase (Mnk) 1 (SEQ ID NO: 5; GenBank Accession
Number AB000409 or NM.sub.--003684 or XM.sub.--001600)
[0131] FIG. 31. Amino Acid sequence of human MAP kinase-interacting
kinase (Mnk) 1 (SEQ ID NO: 6; GenBank Accession Number AB000409 or
NM.sub.--003684 or XM.sub.--001600)
[0132] FIG. 4 shows the eye phenotype induced by overexpression of
an uncoupling protein (dUCPy) that was used to discover factors
modulating uncoupling protein activity. In the fly shown in the
left part of the picture, dUCPy is expressed at normal levels. In
the fly shown in the right part of the photograph, dUCPy is
overexpressed, and the eye is reduced.
[0133] FIG. 5 shows the expression of the Mnk2 gene.
[0134] FIG. 5A shows the real-time PCR analysis of Mnk2 in wildtype
mouse tissues
[0135] FIG. 5B shows the expression of mouse Mnk2 gene in fasted
and obese (ob/ob) mice
[0136] FIG. 5C shows the expression of mouse Mnk2 gene in fasted
and obese mice
[0137] FIG. 5D shows the real-time PCR mediated comparison of Mnk2
expression during differentiation of mammalian fibroblast (3T3-L1)
cells from pre-adipocytes to mature adipocytes
[0138] FIG. 5E shows real-time PCR mediated comparison of Mnk2
expression during the differentiation of mammalian fibroblast
3T3-F442A cells from preadipocytes to mature adipocytes
[0139] FIG. 5F shows real-time PCR mediated comparison of Mnk2
expression during the differentiation of mammalian fibroblast TA1
cells from preadipocytes to mature adipocytes
[0140] FIG. 6 shows the expression of the mouse Mnk1 gene.
[0141] FIG. 6A shows the real-time PCR analysis of Mnk1 in wildtype
mouse tissues
[0142] FIG. 6B shows the real-time PCR mediated comparison of Mnk1
expression in different mouse models
[0143] FIG. 6C shows the real-time PCR mediated comparison of Mnk1
expression during differentiation of mammalian fibroblast 3T3-L1
cells from pre-adipocytes to mature adipocytes
[0144] FIG. 6D shows real-time PCR mediated comparison of Mnk1
expression during the differentiation of mammalian fibroblast
3T3-F442A cells from preadipocytes to mature adipocytes
[0145] FIG. 6E shows real-time PCR mediated comparison of Mnk1
expression during the differentiation of mammalian fibroblast TA1
cells from preadipocytes to mature adipocytes
[0146] FIG. 7 shows the UCPy sequences
[0147] FIG. 7A shows the nucleic acid sequence encoding the
Drosophila UCPy protein (SEQ ID NO. 7). The open reading frame is
underlined.
[0148] FIG. 7B shows the amino acid sequence encoding the
Drosophila UCPy protein (SEQ ID NO. 8).
[0149] FIG. 8: In vitro assays for the determination of
triglyceride storage, synthesis and transport.
[0150] FIG. 8A shows reduction in cellular triglyceride levels
(.mu.g/mg protein) in cells over expressing Mnk2 compared to
control cells. All samples were analysed in duplicates (s1; sample
1, s2; sample 2). The Y-axis shows cellular triglyceride levels
(.mu.g/mg protein) and the X-axis shows days of cell
differentiation.
[0151] FIG. 8B shows reduction in insulin stimulated lipid
synthesis (dpm/mg protein) in cells over expressing Mnk2 compared
to control cells. All samples were analysed in duplicates (s1;
sample 1, s2; sample 2). CB; cytochalasin B, illustrates the
background synthesis in 3T3L1, O; represents the baseline or
un-stimulated glucose transport and hence basal lipid synthesis in
the cells, while Ins; insulin shows the stimulated glucose
transport and the consequent synthesis of glucose to lipid in 3T3L1
cells. The Y-axis displays disintegrations per minutes/mg protein
(dpm/mg protein) and the X-axis denotes the aforementioned
proteins.
[0152] FIG. 8C shows reduction in active transport (AT) of free
fatty acids across the plasma membrane of cells over expressing
Mnk2 compared to control cells. All samples were analysed in
duplicates (as illustrated by twin bar of identical shadings). PD;
passive diffusion illustrated the baseline or non-energy dependant
transportation of exogenous fatty acids across the membrane. AT;
active transport represents energy dependant transportation of
fatty acids across the membrane. The Y-axis shows disintegrations
per minutes/mg protein (dpm/mg protein) and the X-axis displays the
afore mentioned proteins.
[0153] FIG. 9 shows the expression of human Mnk2 in different human
tissues.
[0154] FIG. 9A shows the expression of human Mnk2A and Mnk2B in
different human tissues.
[0155] FIG. 9B shows the expression of human Mnk2A and human Mnk2B
during adipocyte differentiation.
[0156] FIG. 10 shows the expression of the ectopic mouse Mnk2
(mMnk2DN) transgene in actin-mMnk2DN transgenic mice.
[0157] Shown is a taqman analysis on different tissues isolated
from male actin-mMnk2DN transgenic mice and male wild-type
littermates. Data are expressed as fold RNA induction relative to
the corresponding wild-type (wt) tissue. The number on top of each
bar indicate the fold induction relative to the corresponding wt
tissue. Shown is a representative experiment.
[0158] FIG. 11 shows that the ectopic mouse Mnk2 (mMnk2DN)
expression in actin-mMnk2DN transgenic mice leads to increased body
weight
[0159] Shown are growth curves from male bactin-mMnk2DN transgenic
mice (dark grey graph) and male wt littermates (light grey graph)
on high fat diet over a time of 10 weeks. Data are expressed as
mean body weight over time .+-.SE. Shown is a representative
experiment with N=8 respectively N=10 mice per group.
[0160] FIG. 12 shows the exon/intron boundaries of the mouse Mnk2
gene Exon/intron boundaries of the mouse Mnk2 gene are illustrated
in this figure. Exon numbers, the position of the exons on the cDNA
(GenBank accesion number BC010256) and intron lengths are
indicated. Intron sequences are shown in lowercase letters, exon
sequences are shown in capital, bold letters.
[0161] FIG. 13 illustrates the targeted deletion of the mouse Mnk2
gene by homologous recombination.
[0162] The top line shows the wild type locus of mouse Mnk2, the
graphic in the middle shows the targeting vector, and the graphic
at the bottom part of the figure illustrates the targeted locus.
The exons are shown as black boxes. Restriction sites, translation
start site, and stop codon are indicated. The PGK-NEO cassette and
the TK cassette are shown as grey boxes. 4.4 kb of genomic region
of the mouse Mnk2 gene is replaced by a PGK NEO cassette. The
deleted region is indicated. The outside flanking probe used for
Southern blot analysis is shown by a black bar. The genomic
fragments detected with this probe on EcoR1 digested DNA are shown
as arrows. See examples for more detail.
[0163] FIG. 14 shows that purified Mnk2a can be activated in vitro
with a preparation of the kinases Erk2 and the double point mutant
Mek1 S218D S222E.
THE EXAMPLES ILLUSTRATE THE INVENTION
Example 1
Measurement of Triglyceride Content of Homozygous Flies (FIG.
1)
[0164] The change of triglyceride content of Drosophila
melanogaster containing a special expression system (EP-element,
Rorth P, Proc Natl Acad Sci USA 1996, 93(22):12418-22) was
measured. Mutant flies are obtained from a fly mutation stock
collection. The flies are grown under standard conditions known to
those skilled in the art, and in the course of the experiment,
additional feedings with bakers yeast (Saccharomyces cerevisiae)
are provided. Specifically, homozygous male EP(3)3333 and EP(3)3576
flies were investigated in comparison to control flies (FIG. 1).
For determination of triglyceride content, flies were incubated for
5 min at 90.degree. C. in an aqueous buffer using a waterbath,
followed by hot extraction. After another 5 min incubation at
90.degree. C. and mild centrifugation, the triglyceride content of
the flies extract was determined using Sigma Triglyceride (INT
336-10 or -20) assay by measuring changes in the optical density
according to the manufacturer's protocol. As a reference protein
content of the same extract was measured using BIO-RAD DC Protein
Assay according to the manufacturer's protocol. The assay was
repeated several times. The-average triglyceride level of EP
collection is shown as 100% in FIG. 1. EP(3)3333 and EP(3)3576
homozygous flies show constantly a higher triglyceride content than
the controls (approx. 140%). Therefore, the change of gene activity
in the locus 86F7 (estimated), where the EP-vector of EP(3)3333 and
EP(3)3576 flies is homozygous viably integrated into the Lk6 gene
locus, is responsible for changes in the metabolism of the energy
storage triglycerides, therefore representing in both cases an
obese fly model.
Example 2
Identification of the Drosophila Gene Responsible for the Change in
the Metabolism of the Energy Storage Triglycerides (FIG. 2)
[0165] Genomic DNA sequences were isolated that are localized
directly adjacent to the integration of the EP vectors (herein
EP(3)3333 and EP(3)3576). Using those isolated genomic sequences,
public databases like Berkeley Drosophila Genome Project (GadFly)
were screened thereby confirming the homozygous viable integration
site of the EP(3)3333 and EP(3)3576 vectors. FIG. 2 shows the
molecular organization of this gene locus. In FIG. 2, genomic DNA
sequence is represented by the assembly as a dotted black line
(from position 7544500 to 7559500 on chromosome 3R) that includes
the integration sites of EP(3)3333 and EP(3)3576. Transcribed DNA
sequences (expressed sequence tags, ESTs) and predicted exons are
shown as bars in the lower two lines. Predicted exons of gene
CG17342 (GadFly, Lk6, homologous to Mnk) are shown as dark grey
bars and introns are shown as slim grey lines in the middle of the
figure. Using plasmid rescue method genomic DNA sequences that are
directly localised 3' of the EP(3)3333 and EP(3)3576 integration
site were isolated. Using the plasmid rescue DNA public DNA
sequence databases were screened thereby identifying the
integration site of EP(3)3333 and EP(3)3576 causing an increase of
triglyceride content. EP(3)3333 is integrated in the 5' region of a
5 prime exon of the gene CG17342 and EP(3)3576 in the 5' region of
an alternative 5' exon. Mnk encodes for a gene that is predicted by
GadFly sequence analysis programs as CG17342. Therefore, expression
of the CG17342 could be affected by homozygous viable integration
of the EP(3)3333 and EP(3)3576 leading to increase of the energy
storage triglycerides and a change of uncoupling protein
activity.
Example 3
Cloning of a Drosophila melanogaster Gene with Homology to Human
Uncoupling Proteins (UCPs) (FIG. 7)
[0166] Sequences homologous to human UCP2 and UCP3 genes were
identified using the publicly available program BLAST of the data
base of the National Center for Biotechnology Information
(NCBI)(see, Altschul et al., 1997, Nucleic Acids Res.
25:3389-3402). The homology search yielded sequence fragments of a
family of Drosophila genes with UCP homology. They are clearly
different to the next related mitochondrial proteins (oxoglutarate
carrier).
[0167] Using the sequence fragment of one of this genes (herein
referred to as dUCPy'), a PCR primer pair was generated (Upper
5'-CTAAACAAACAATTCCAAACATAG (SEQ ID NO: 9), Lower 5
prime-AAAAGACATAGAAAATACGATAGT (SEQ ID NO: 10)) and a PCR reaction
performed on Drosophila cDNA using standard PCR conditions. The
amplification product was radioactively labelled and used to screen
a cDNA library prepared from adult Drosophila flies (Stratagene). A
full-length cDNA clone was isolated, sequenced (FIG. 7), and used
for further experiments. The nucleotide sequence of dUCPy is shown
in FIG. 7A (SEQ ID NO: 7), the deduced open reading is shown in
FIG. 7B (SEQ ID NO: 8).
Example 4
Cloning of the dUCPy cDNA into an Drosophila Expression Vector
[0168] In order to test the effects of dUCPy expression in
Drosophila cells, the dUCPy cDNA was cloned into the expression
vector pUAST (Brand A & Perrimon N, Development 1993,
118:401-415) using the restriction sites NotI and KpnI. The
resulting expression construct was injected into the germline of
Drosophila embryos and Drosophila strains with a stable integration
of the construct were generated. Since the expression vector pUAST
is activated by the yeast transcription factor Gal4 which is
normally absent from Drosophila cells dUCPy is not yet expressed in
these transgenic animals. If pUAST-dUCPy flies are crossed with a
second Drosophila strain that expresses Gal4 in a tissue specific
manner the offspring flies of this mating will express dUCPy in the
Gal4 expressing tissue.
[0169] The cross of pUAST-dUCPy flies with a strain that expresses
Gal4 in all cells of the body (under control of the actin promoter)
showed no viable offspring. This means that dUCPy overexpression in
all body cells is lethal. This finding is consistent with the
assumption that dUCPy overexpression could lead to a collapse of
the cellular energy production.
[0170] Expression of dUCPy in a non-vital organ like the eye (Gal4
under control of the eye-specific promoter of the "eyeless" gene)
results in flies with visibly damaged eyes. This easily visible eye
phenotype is the basis of a genetic screen for gene products that
can modify UCP activity.
Example 5
dUCPy Modifier Screen (FIG. 4)
[0171] Parts of the genomes of the strain with Gal4 expression in
the eye and the strain carrying the pUAST-dUCPy construct were
combined on one chromosome using genomic recombination. The
resulting fly strain has eyes that are permanently damaged by dUCPy
expression. Flies of this strain were crossed with flies of a large
collection of mutagenized fly strains. In this mutant collection a
special expression system (EP-element, see Rorth, 1996, supra) is
integrated randomly in different genomic loci. The yeast
transcription factor Gal4 can bind to the EP-element and activate
the transcription of endogenous genes close the integration site of
the EP-element. The activation of the genes therefore occurs in the
same cells (eye) that overexpress dUCPy. Since the mutant
collection contains several thousand strains with different
integration sites of the EP-element it is possible to test a large
number of genes whether their expression interacts with dUCPy
activity. In case a gene acts as an enhancer of UCP activity the
eye defect will be worsened; a suppressor will ameliorate the
defect (see FIG. 4).
[0172] Using this screen a gene with enhancing activity was
discovered that was found to be the LK6 kinase in Drosophila.
Example 6
Cloning of Lk6 from Drosophila (FIG. 3A)
[0173] Genomic DNA neighbouring to the eye-defect rescuing
EP-element was cloned by inverse PCR and sequenced. This sequence
was used for a BLAST search in a public Drosophila gene database.
The amino acid sequence of the Drosophila protein is shown in FIG.
3A (referred to as dmAAB18789).
Example 7
Identification of Mammalian LK6 Homologous Proteins (FIG. 3)
[0174] Sequences homologous to Drosophila Lk6 were identified using
the publicly available program BLASTP 2.2.3 of the non-redundant
protein data base of the National Center for Biotechnology
Information (NCBI)(see, Altschul et al., 1997, Nucleic Acids Res.
25:3389-3402).
[0175] Mnk homologous proteins and nucleic-acid molecules coding
therefore are obtainable from insect or vertebrate species, e.g.
mammals or birds. Particularly preferred are human Mnk homologous
polypeptides and nucleic acids, particularly polypeptides and
nucleic acids encoding a human Mnk2 protein (Genbank Accession No.
AF237775 and NM.sub.--017572.1; Genbank Accession No. AF237775 is
identical to formerly Genbank Accession No. XM.sub.--030637 which
was removed at the submitters request; see a Clustal W multiple
sequence alignment in FIG. 3B) or nucleic acids encoding a human
Mnk1 protein (Genbank Accession No. AB000409.1 and
NM.sub.--003684.2; Genbank Accession No. AB000409.1 is identical to
formerly Genbank Accession No. XM.sub.--001 600 which was removed
at the submitters request; see a Clustal W multiple sequence
alignment in FIG. 3C).
[0176] FIG. 3A shows the alignment of the Mnk proteins from
different species, hXP.sub.--030637 refers to human Mnk2 (identical
to Genbank Accession No. AF237775), hXP.sub.--001600 refers to
human Mnk1 (identical to Genbank Accession No. AB000409.1), and
dmAB18789 refers to the protein encoded by Drosophila gene with
GadFly Accession No. CG17342.
[0177] The mouse homologous polypeptides of the invention were
identified as GenBank Accession Numbers NP.sub.--067437.1 (for the
mouse homolog MAP kinase-interacting serine/threonine kinase 2;
Mnk2; for the cDNA GenBank Accession Number' BC010256) and GenBank
Accession Numbers NP.sub.--067436.1 (for the mouse homolog MAP
kinase-interacting serine/threonine kinase 1; Mnk1).
Example 8
Expression of the Polypeptides in Mammalian (Mouse) Tissues (FIG. 5
and FIG. 6)
[0178] For analyzing the expression of the polypeptides disclosed
in this invention in mammalian tissues, several mouse strains
(preferably mice strains C57BI/6J, C57BI/6 ob/ob and C57BI/KS db/db
which are standard model systems in obesity and diabetes research)
were purchased from Harlan Winkelmann (33178 Borchen, Germany) and
maintained under constant temperature (preferably 22.degree. C.),
40 per cent humidity and a light/dark cycle of preferably 14/10
hours. The mice were fed a standard chow (for example, from ssniff
Spezialitten GmbH, order number ssniff M-Z V1126-000). For the
fasting experiment ("fasted wild type mice"), wild type mice were
starved for 48 h without food, but only water supplied ad libitum.
(see, for example, Schnetzler et al. J Clin Invest 1993
July;92(1):272-80, Mizuno et al. Proc Natl Acad Sci USA 1996 April
16;93(8):3434-8). Animals were sacrificed at an age of 6 to 8
weeks. The animal tissues were isolated according to standard
procedures known to those skilled in the art, snap frozen in liquid
nitrogen and stored at -80.degree. C. until needed.
[0179] For analyzing the role of the proteins disclosed in this
invention in the in vitro differentiation of different mammalian
cell culture cells for the conversion of pre-adipocytes to
adipocytes, mammalian fibroblast (3T3-L1) cells (e.g., Green &
Kehinde, Cell 1: 113-116, 1974) were obtained from the American
Tissue Culture Collection (ATCC, Hanassas, Va., USA; ATCC-CL 173).
3T3-L1 cells were maintained as fibroblasts and differentiated into
adipocytes as described in the prior art (e.g., Qiu. et al., J.
Biol. Chem. 276:11988-95, 2001; Slieker et al., BBRC 251: 225-9,
1998). In brief, cells were plated in DMEM/10% FCS (Invitrogen,
Karlsruhe, Germany) at 50,000 cells/well in duplicates in 6-well
plastic dishes and cultured in a humidified atmosphere of 5%
CO.sub.2 at 37.degree. C. At confluence (defined as day 0: d0)
cells were transferred to serum-free (SF) medium, containing
DMEM/HamF12 (3:1; Invitrogen), Fetuin (300 .mu.g/ml; Sigma, Munich,
Germany), Transferrin (2 .mu.g/ml; Sigma), Pantothenate (17 .mu.M;
Sigma), Biotin (1 .mu.M; Sigma), and EGF (0.8 nM; Hoffmann-La
Roche, Basel, Switzerland). Differentiation was induced by adding
Dexamethasone (DEX; 1 .mu.M; Sigma),
3-Methyl-Isobutyl-1-Methylxahthine (MIX; 0.5 mM; Sigma), and bovine
Insulin (5 .mu.g/ml; Invitrogen). Four days-after confluence (d4),
cells were kept in SF medium, containing bovine Insulin (5
.mu.g/ml) until differentiation was completed. At various time
points of the differentiation procedure, beginning with day 0 (day
of confluence) and day 2 (hormone addition; for example,
dexamethason and 3-isobutyl-1-methylxanthin), up to 10 days of
differentiation, suitable aliquots of cells were taken every two
days.
[0180] Alternatively, mammalian fibroblast 3T3-F442A cells (e.g.,
Green & Kehinde, Cell 7: 105-113, 1976) were obtained from the
Harvard Medical School, Department of Cell Biology (Boston, Mass.,
USA). 3T3-F442A cells were maintained as fibroblasts and
differentiated into adipocytes as described previously (Djian, P.
et al., J. Cell. Physiol., 124:554-556, 1985). At various time
points of the differentiation procedure, beginning with day 0 (day
of confluence and hormone addition, for example, Insulin), up to 10
days of differentiation, suitable aliquots of cells were taken
every two days. 3T3-F442A cells are differentiating in vitro
already in the confluent stage after hormone (insulin)
addition.
[0181] TaqMan Analysis of the proteins of the invention was carried
out (FIG. 5 and FIG. 6). RNA was isolated from mouse tissues or
cell culture cells using Trizol Reagent (for example, from
Invitrogen, Karlsruhe, Germany) and further purified with the
RNeasy Kit (for example, from Qiagen, Germany) in combination with
an DNase-treatment according to the instructions of the
manufacturers and as known to those skilled in the art. Total RNA
was reverse transcribed (preferably using Superscript II
RNaseH-Reverse Transcriptase, from Invitrogen, Karlsruhe, Germany)
and subjected to Taqman analysis preferably using the Taqman
2.times.PCR Master Mix (from Applied Biosystems, Weiterstadt,
Germany; the Mix contains according to the Manufacturer for example
AmpliTaq Gold DNA Polymerase, AmpErase UNG, dNTPs with dUTP,
passive reference Rox and optimized buffer components) on a GeneAmp
5700 Sequence Detection System (from Applied Biosystems,
Weiterstadt, Germany).
[0182] For the analysis of the expression of Mnk2 or Mnk1, taqman
analysis was performed using the following primer/probe pairs:
1 (Seq ID NO: 11) Mouse Mnk1 forward primer 5'-GCT GAG GGC CTC TGC
TCC-3'; (Seq ID NO: 12) Mouse Mnk1 reverse primer 5'-TCG CCT TCG
AGC GAG G-3'; (Seq ID NO: 13) Mouse Mnk1 Taqman probe (5/6-FAM) TGA
AGC TGT CCC CTC CAT CCA AAT CTC (5/6-TAMRA) (SEQ ID NO: 14)
Taqman-1856F Mnk2 forward primer: 5'-TGCACTTGATTGACCCCGA-3' (SEQ ID
NO: 15) Taqman-1923R Mnk2 reverse primer:
5'-TTTGTGATTGTCAACCCTCCAA-3' (SEQ ID NO: 16) Taqman-1877T Mnk2
Taqman probe: (5/6-FAM)-CCCCATCATCCACCTGCAGTGTCC-(5/6TAMRA)
[0183] Taqman analysis revealed that Mnk2 is the more interesting
homologue of the fly Lk6 gene. The results are shown in FIG. 5 and
FIG. 6. In comparison to Mnk1, which is rather ubiquitously
expressed, Mnk2 shows its highest expression levels in the brown
and white adipose tissues (FIGS. 5A and 6A, respectively). The
expression of Mnk2 in white adipose tissue is under metabolic
control: In fasted as well as obese (ob/ob) mice, expression is
reduced to about 40% of wildtype levels (FIG. 5C; see also FIG.
5B). In addition, expression of Mnk2 is strongly induced during the
in vitro differentiation of 3T3-L1 (FIG. 5D) as well as of two
additional model systems for the in vitro differentiation of
preadipocytes to adipocytes, the 3T3-F442A and TA1 cell lines (FIG.
5E and FIG. 5F, respectively). Contrary to this, the relative
expression levels of Mnk1 remain unchanged during the
differentiation of these cell lines (FIGS. 6D and 6E,
respectively).
Example 9
Expression of the Polypeptides in Mammalian (Human) Tissues (FIG.
9)
[0184] Human primary adipocytes were differentiated into mature
adipocytes as described by Hauner et al. 1989 (J Clin
Invest84(5):1663-70). Briefly, cells were grown in DMEM/Nutrient
Mix F12, 1% PenStrep, 17 .mu.M Biotin, 33 .mu.M Pantothenat, 10%
none heat inactivated fetal calf serum. On day 0 of
differentiation, the medium was changed to DMEM/Nutrient Mix F12,
1% Pen/Strep, 17 .mu.M Biotin, 33 .mu.M Pantothenat, 0,01 mg/ml
Transferrin, Hydrocortisone, 20 nM humanes Insulin, 0,2 nM T3, 25
nM Dexamethasone, 250 .mu.M IBMX, 3 .mu.M Rosiglitazone. On day 4
of differentiation, the medium was changed to DMEM/Nutrient Mix F12
1% Pen/Strep, 17 .mu.M Biotin, 33 .mu.M Pantothenat, 0,01 mg/ml
Transferrin, 100 nM Hydrocortisone, 20 nM humanes Insulin, 0,2 nM
T3. At various time points of the differentiation procedure,
beginning with day 0 (day of confluence) and day 4 (hormone
addition), up to 14 days of differentiation, suitable aliquots of
cells were taken every two days. RNA was isolated from human cell
culture cells using Trizol Reagent (for example, from Invitrogen,
Karlsruhe, Germany) and further purified with the RNeasy Kit (for
example, from Qiagen, Germany) in combination with an
DNase-treatment according to the instructions of the' manufacturers
and as known to those skilled in the art.
[0185] In addition to the RNA isolated from human adipocytes at
different differentiation stage, RNAs isolated from different human
tissues were obtained from Invitrogen Corp., Karlsruhe, Germany:
(i) total RNA from human adult skeletal muscle (Invitrogen Corp.
Order Number 735030); (ii) total RNA from human adult lung
(Invitrogen Corp. Order Number 735020); (iii) total RNA from human
adult liver (Invitrogen Corp. Order Number 735018); (iv) total RNA
from human adult placenta (Invitrogen Corp. Order Number 735026);
(v) total RNA from human adult testis (Invitrogen Corp. Order
Number 64101-1); (vi) total RNA from human normal adipose tissue
(Invitrogen Corp. Order Number D6005-01); (vii) total RNA from
human normal pancreas (Invitrogen Corp. Order Number DG6101);
(viii)total RNA from human normal brain (Invitrogen Corp. Order
Number D6030-01). The RNA was treated with DNase according to the
instructions of the manufacturers (for example, from Qiagen,
Germany) and as known to those skilled in the art.
[0186] Total RNA was reverse transcribed (preferably using
Superscript II RNaseH-Reverse Transcriptase, from Invitrogen,
Karlsruhe, Germany) and subjected to Taqman analysis preferably
using the Taqman 2.times.PCR Master Mix' (page 66, line 31:
Weiterstadt, Germany; see Example 8).
[0187] Taqman analysis was performed preferably using the following
primer/probe pairs:
[0188] For the amplification of human Mnk2a:
[0189] human Mnk2a forward primer (SEQ ID NO: 17): 5'-cca tot ccc
cot ctg tac ata gg-3'; human Mnk2a reverse primer (SEQ ID NO: 18):
5'-cog got ggc gat agc tta a-3'; Taqman probe (SEQ ID NO: 19):
(5/6-FAM) cac cog tcc ccc aat caa atc taa agg (5/6-TAMRA)
[0190] For the amplification of human Mnk2b:
[0191] human Mnk2b forward primer (SEQ ID NO: 20): 5'-TTA CTG TGA
ATG AGT GAA GAT CCT GG-3'; human Mhk2b reverse primer (SEQ ID NO:
21): 5'-ATG GCC GTT CAC CGT CC-3'; Taqman probe (SEQ ID NO: 22):
(5/6-FAM) CCA GGC CAG CTC CCA TCG CTG (5/6-TAMRA)
[0192] As shown in FIG. 9A, real time PCR (Taqman) analysis of the
expression of Mnk2a and Mnk2b protein in human tissues revealed
that both proteins are expressed in all tissues analysed with high
levels of expression in adipose tissue, muscle, lung, testis, and
placenta.
[0193] The relative expression levels of both human Mnk2 splice
variants is the same for all tissues analysed. Both show highest
expression levels in tissues relevant for metabolic disorders
namely adipose and muscle tissue.
[0194] As shown in FIG. 9B, Mnk2a as well as Mnk2b are upregulated
during human adipocyte differentiation. This suggests a function of
both proteins in the metabolism of mature adipocytes.
Example 10
Assays for the Determination of Triglyceride Storage, Synthesis and
Transport (FIG. 8)
[0195] Retroviral Infection of Preadipocytes
[0196] Packaging cells were transfected with retroviral plasmids
pLPCX carrying mouse Mnk2 transgene and a selection marker using
calcium phosphate procedure. Control cells were infected with pLPCX
carrying no transgene.
[0197] Briefly, exponentially growing packaging cells were seeded
at a density of 350,000 cells per 6-well in 2 ml DMEM+10% FCS one
day before transfection. 10 min before transfection chloroquine was
added directly to the overlying medium (25 .mu.M final
concentration). A 250 .mu.l transfection mix consisting of 5 .mu.g
plasmid-DNA (candidate:helper-virus in a 1:1 ratio) and 250 mM
CaCl.sub.2 was prepared in a 15 ml plastic tube. The same volume of
2.times.HBS (280.mu.M NaCl, 50.mu.M HEPES, 1.5 mM
Na.sub.2HPO.sub.4, pH 7.06) was added and air bubbles were injected
into the mixture for 15 sec. The transfection mix was added drop
wise to the packaging cells, distributed and the cells were
incubated at 37.degree. C., 5% CO.sub.2 for 6 hours. The cells were
washed with PBS and the medium was exchanged with 2 ml DMEM+10% CS
per 6-well. One day after transfection the cells were washed again
and incubated for 2 days of virus collection in 1 ml DMEM+10% CS
per 6-well at 32.degree. C., 5% CO2. The supernatant was then
filtered through a 0.45 .mu.m cellulose acetate filter and
polybrene (final concentration 8 .mu.g/ml) was added. Mammalian
fibroblast (3T3-L1) cells in a sub-confluent state were overlaid
with the prepared virus containing medium. The infected cells were
selected for 1 week with 2.mu.g/ml puromycin. Following selection
the cells were checked for transgene expression by western blot and
immunofluorescence. Over expressing cells were seeded for
differentiation.
[0198] 3T3-L1 cells were maintained as fibroblasts and
differentiated into adipocytes as described in the prior art and in
example 8. For analysing the role of the proteins disclosed in this
invention in the in vitro assays for the determination of
triglyceride storage, synthesis and transport were performed.
[0199] Preparation of Cell Lysates for Analysis of Metabolites
[0200] Starting at confluence (DO), cell media was changed every 48
hours. Cells and media were harvested 8 hours prior to media change
as follows. Media was collected, and cells were washed twice in PBS
prior to lyses in 600 .mu.l HB-buffer (0.5% Polyoxyethylene 10
tridecylethan, 1 mM EDTA, 0.01M NaH.sub.2PO.sub.4, pH 7.4). After
inactivation at 70.degree. C. for 5 minutes, cell lysates were
prepared on Bio 101 systems lysing matrix B (0.1 mm silica beads;
Q-Biogene, Carlsbad, USA) by agitation for 2.times.45 seconds at a
speed of 4.5 (Fastprep FP120, Bio 101 Thermosavant, Holbrock, USA).
Supernatants of lysed cells were collected after centrifugation at
3000 rpm for 2 minutes, and stored in aliquots for later analysis
at -80.degree. C.
[0201] Changes in Cellular Triglyceride Levels During Adipogenesis
(FIG. 8A)
[0202] Cell lysates and media were simultaneously analysed in
96-well plates for total protein and triglyceride content using the
Bio-Rad DC Protein assay reagent (Bio-Rad, Munich, Germany)
according to the manufacturer's instructions and a modified
enzymatic triglyceride kit (GPO-Trinder; Sigma) briefly final
volumes of reagents were adjusted to the 96-well format as follows:
10 .mu.l sample was incubated with 200 .mu.l reagent A for 5
minutes at 37.degree. C. After determination of glycerol (initial
absorbance at 540 nm), 50 .mu.l reagent B was added followed by
another incubation for 5 minutes at 37.degree. C. (final absorbance
at 540 nm). Glycerol and triglyceride concentrations were
calculated using a glycerol standard set (Sigma) for the standard
curve included in each assay.
[0203] As shown in FIG. 8A, we found that in Mnk2 overexpressing
cells cellular triglyceride levels were significantly lower from
day 4 to day 12 of adipogenesis compared to that in the control
cells (FIG. 8A). These results indicate that Mnk2 targets
regulatory pathways or enzymes involved in lipid metabolism, which
we analysed in more detail in the lipid synthesis and FFA transport
assays described-below.
[0204] Synthesis of Lipids During Adipogenesis (FIG. 8B)
[0205] During the terminal stage of adipogenesis (day 12) cells
were analysed for their ability to metabolise lipids. A modified
protocol to the method of Jensen et al (2000), JBC 275, 40148, for
lipid synthesis was established. Cells were washed 3 times with PBS
prior to serum starvation in Krebs-Ringer-Bicarbonate-Hepes buffer
(KRBH; 134 nM NaCl, 3.5 mM KCl, 1.2 mM KH.sub.2 PO.sub.4, 0.5 mM
MgSO.sub.4, 1.5 mM CaCl.sub.2, 5 mM NaHCO.sub.3, 10 mM Hepes, pH
7.4), supplemented with 0.1% FCS for 2.5 h at 37.degree. C. For
insulin-stimulated lipid synthesis, cells were incubated with 1
.mu.M bovine insulin (Sigma; carrier: 0.005 N HCl) for 45 min at
37.degree. C. Basal lipid synthesis was determined with carrier
only. .sup.14C(U)-D-Glucose (NEN Life Sciences) in a final activity
of 1 .mu.Ci/Well/ml in the presence of 5 mM glucose was added for
30 min at 37.degree. C. For the calculation of background
radioactivity, 25 .mu.M Cytochalasin B (Sigma) was used. All assays
were performed in duplicate wells. To terminate the reaction, cells
were washed 3 times with ice cold PBS, and lysed in 1 ml 0.1 N
NaOH. Protein ) concentration of each well was assessed using the
standard Biuret method (Protein assay reagent; Bio-Rad). Total
lipids were separated from aqueous phase after overnight extraction
in Insta-Fluor scintillation cocktail (Packard Bioscience) followed
by scintillation counting.
[0206] Our results clearly show that Mnk2 overexpressing cells were
less effective at synthesising lipids from exogenous glucose.
Consequently, the levels of insulin stimulated lipid synthesis are
significantly lower at day 12 of adipogenesis when compared to
control cells (FIG. 8B). The lower lipid levels observed in the
experiments above therefore result most likely from a lower lipid
synthesis rate and are not the result of an increased turn over of
lipid stores.
[0207] Transport and Metabolism of Free Fatty Acids Across During
Adipogenesis (FIG. 8C)
[0208] During the terminal stage of adipogenesis (D12) cells were
analysed for their ability to transport long chain fatty acid
across the plasma membrane. A modified protocol to the method of
Abumrad et al (1991)(Proc. Natl. Acad. Sci. USA, 1991: 88; 6008-12)
for cellular transportation of fatty acid was established. In
summary, cells were washed 3 times with PBS prior to serum
starvation. This was followed by incubation in KRBH buffer,
supplemented with 0.1% FCS for 2.5 h at 37.degree. C. Uptake of
exogenous free fatty acids was initiated by the addition of
isotopic media containing non radioactive oleate and
(.sup.3H)oleate (NEN Life Sciences) complexed to serum albumin in a
final activity of 1 .mu.Ci/Well/ml in the presence of 5 mM glucose
for 30 min at room temperature (RT). For the calculation of passive
diffusion (PD) in the absence of active transport (AT) across the
plasma membrane 20 mM of phloretin in glucose free media (Sigma)
was added for 30 min at room temperature (RT). All assays were
performed in duplicate wells. To terminate the active transport 20
mM of phloretin in glucose free media was added to the cells. Cells
were lysed in 1 ml 0.1 N NaOH and the protein concentration of each
well were assessed using the standard Biuret method (Protein assay
reagent; Bio-Rad). Esterified fatty acids were separated from free
fatty acids using overnight extraction in Insta-Fluor scintillation
cocktail (Packard Bioscience) followed by scintillation
counting.
[0209] We found that transport of exogenous fatty acids across the
plasma membrane of Mnk2 overexpressing cells and hence
esterification of these metabolites were considerably lower at day
12 of adipogenesis when compared to control cells (FIG. 8C). Taken
together the overexpression of Mnk2 showed an effect on
triglyceride metabolism in all three assays we performed in 3T3-L1
cells, making it a potential interesting drug target to treat
metabolic disorders.
Example 11
Generation and Analysis of Mnk2 Transgenic Animals
(.beta.-Actin-mMnk2DN)
[0210] Generation of the Transgenic Animals
[0211] Mouse Mnk2 cDNA was isolated from mouse brown adipose tissue
(BAT) using standard protocols as known to those skilled in the
art. The cDNA was amplified by RT-PCR using the following primer
pair:
2 (SEQ ID NO: 23) Mnk2 forward primer: 5' AAG TTG GCC TTC GCG TTA
GAC 3' (SEQ ID NO: 24) Mnk2 reverse primer: 5' CGA TAT GTA CAA GGA
GCT AG 3'.
[0212] The resulting Mnk2 cDNA was cloned into pBluescript KS+
(Stratagene) according to standard protocols, resulting in a
plasmid referred to as pKS+-mMnk2'. The cDNA of pKS+-mMnk2 c was
mutated using site directed mutatagenesis (Stratagene), according
to the manufacturer's instructions. Using the Mnk2 top oligo (SEQ
ID NO: 25): 5' CTC CCC CAT CTC CGC ACC AGA GCT GCT CGC CCC GTG TGG
GTC AG 3' and the Mnk2 bottom oligo (SEQ ID NO: 26): 5' CTG ACC CAC
ACG GGG CGA GCT CTG GTG CGG AGA TGG GGG AG 3', two point mutations
were introduced into the cDNA resulting in amino acid exchanges at
position T197 and T202 to A197 and A202 of the Mnk2 cDNA.
[0213] The resulting mutated cDNA (referred to as mMnk2DN) was
cloned into the EcoRV cloning site of the transgenic expression
vector pTG-.beta.-actin-X-hgh-bgh-polyA. The .beta.-actin-Mnk2DN
transgene was microinjected into the male pronucleus of fertilized
mouse embryos (preferably strain C57/BL6/CBA F1 (Harlan
Winkelmann). Injected embryos were transferred into pseudo-pregnant
foster mice. Transgenic founders were detected by PCR analysis
using the forward primer (SEQ ID NO: 27): 5' GCT GCT GGT CCG AGA
TGC C 3' and reverse primer (SEQ ID NO: 28): 5' GGG TCA TGC GCG ATC
CCC 3'. Two independent transgenic mouse lines containing the
.beta.-actin-Mnk2DN construct were established and kept on a
C57/BL6 background. Briefly, founder animals were backcrossed with
C57/BL6 mice to generate F1 mice for analysis. Transgenic mice were
continously bred onto the C57/BI6 background.
[0214] Expression of the Construct in Different Mouse Tissues (FIG.
10)
[0215] using standard techniques, .beta.-actin-Mnk2DN transgene
expression was verified by Taqman analysis using forward primer
(SEQ ID NO: 29): 5' CAG CGT GGT AGT ACA GGA CGT G 3', reverse
primer (SEQ ID NO: 30): 5' TCC CTG TGG GCG ATG C 3' and primer (SEQ
ID NO: 31): 5' CAG TGC CCT GGA CTT CCT GCA TAA CAA 3'. Taqman
analysis was performed using a representative panel of mouse
tissues.
[0216] The expression of the bactin-Mnk2DN transgene was observed
in the following tissues: WAT, muscle, liver, kidney, thymus,
heart, lung, and spleen. Expression levels of the transgene were
2.8-16.9 fold increased relative to Mnk2 expression in wild-type
mice, depending on the tissue analyzed. No Mnk2 transgene
expression was detected in BAT tissue (see FIG. 10).
[0217] Analysis of the Bodyweight of the Transgenic Mice (FIG.
11)
[0218] After weaning, male .beta.-actin-mMnk2DN transgenic mice and
their wild-type (wt) littermates-controls were placed in groups of
4 to 5 animals (N=4 up to N=5) on control diet (preferably Altromin
C1057 mod control, 4.5% crude fat or high fat diet (preferably
Altromin C1057mod. high fat, 23.5% crude fat). Total body weight of
the animals was measured weekly over a period of 12-16 weeks.
[0219] On each diet, mean bodyweight of .beta.-actin-mMnk2DN
transgenic mice was clearly increased compared to wildtype
littermates on the respective diet. Significant differences in mean
bodyweight were first observed around the end of postnatal week 4
on both diets. After 10 weeks on high fat diet, the mean bodyweight
of .beta.-actin-mMnk2DN transgenic mice compared to wt littermates
was increased by 8.8 g (=23% increase in mean bodyweight relative
to wt littermates) (FIG. 11). Similar differences in mean body
weight were observed in wt and .beta.-actin-mMnk2DN transgenic mice
on control diet (data not shown). Thus, our results clearly show
that the ectopic expression of mMnk2DN transgene leads to an
increase in bodyweight. The effect appears independently of the
diet give, as it can be seen on control diet as well as on high fat
diet.
Example 11
Generation and Analysis of mMnk2.+-.Mice (FIG. 12 and FIG. 13)
[0220] A 605 base pair probe of the mMnk2 cDNA (GenBank accesion
number BC010256; position 61-665) was amplified from mouse white
adipose tissue (WAT) cDNA by PCR using forward primer (SEQ ID NO:
32): 5' ACA TCA GCC CAC AGT GTG A 3' and reverse primer (SEQ ID NO:
33): 5' TCT CCA TTG AGT TTG ATA CCA 3'. This probe was used to
screen a 129SVJ genomic phage library (obtained from Stratagene).
Three independent clones were isolated and subcloned into the NotI
cloning site of pBluescript KS+ (Stratagene). These genomic clones
were used for restriction mapping and sequencing to characterize
the genomic locus of mouse Mnk2 (FIG. 12). A PGK-neomycin cassette
was inserted into the locus of mouse Mnk2 replacing 4,4 kb of
genomic DNA thereby deleting the complete coding region of mMnk2.
Briefly, an 8kb SpeI-NotI fragment was cloned into the XbaI site of
pBluescript KS+ upstream of the PGK-Neomycin cassette, which was
inserted into the SmaI site of pBluescript. A 1 kb genomic fragment
was amplified by PCR using the following primer pair (non priming
nucleotides/attached restriction sites are lower case letters):
Mnk2-SA forward primer (SEQ ID NO: 34): EcoRI 5' cgg aat CCA CTA
GCT CCT TGT ACA TAT 3'; Mnk2-SA reverse primer (SEQ ID NO: 35):
ClaI 5' cca tcg atG GAA CTC GTA TTG CAT AGT AG 3'. The resulting
fragment was inserted into the EcoRI/ClaI site of pBluescript KS+.
As a negative selection marker a thymidine kinase cassette was
cloned into ClaI/XhoI site of the targeting construct. (FIG. 13)
The construct was linearized by NotI digestion and electropbrated
into mouse embryonic stem (ES) cells. ES cell clones were selected
by G418 and Gancyclovir treatment (preferably 350 .mu.g G418/ml and
2 .mu.m Gancyclovir). Out of 600 neomycin resistant colonies, two
independent homologous recombined ES cell clones were identified by
PCR. The results were confirmed by southern blot analysis with
EcoRI digested genomic DNA using a 3' flanking probe (position
2495-3065 mMnk2 cDNA). A single integration event was confirmed by
Southern blot analysis of BamHI digested DNA with a Neomycin probe.
ES cell clones were aggregated with 8-cell-stage embryos from NMRI
mice and developing blastocysts were transferred into
pseudo-pregnant mice to generate chimeras. Chimeras were bred with
C57/BL6 mice and offsprings were genotyped by PCR using the
following primers: Mnk2-ES primer (SEQ ID NO: 36): 5' AGA CTA GGG
AGG AGG GTG GAG GA 3'; Mnk2-KO primer (SEQ ID NO: 37): 5' GGT GGA
TGT GGA ATG TGT GCG A 3'; Mnk2-WT 5' GGG GTG TAG GGG TCT GTT AGG
3'. Heterozygous mice were used for further intercrosses and
analyzed.
Example 11
Small Molecule Screening
[0221] Compounds which are suitable for the prophylaxis, treatment
or diagnosis of Mnk-related metabolic disorders may be identified
via a kinase assay, a binding assay or any other suitable assay to
measure a function associated with the Mnk polypeptide, a Mnk
polypeptide fragment or derivative thereof. This kinase assay may
be based on recombinant human Mnk2 (Mnk2a or Mnk2b) or Mnk1 protein
and a labelled peptide comprising the eIF4E target sequence, a
labelled recombinant eIF4E target sequence or a labelled
recombinant eIF4E protein as a substrate. The assay may be a
radioactive kinase assay or an assay based on using an
anti-phosphoserine antibody which is capable of recognizing eIF4E
phosphorylation at Ser209.
[0222] For example, the kinases Mnk2a (GenBank Accession Number
AF237775; see also FIGS. 3D and 3E), Erk2 (GenBank Accession Number
M84489) and a double point mutant of Mek1 (GenBank Accession Number
Q02750) containing the amino acid substitutions Ser218Asp and
Ser222Glu (S218D S222E) were expressed in E. coli and subsequently
purified using methods known to those skilled-in the art.
Preferably, in a kinase reaction of 50 .mu.l, 2.0 .mu.M Mnk2a was
incubated with 200 nM Erk2 plus 20 nM Mek1 S218D S222E (labelled
lanes 1 to 4 in FIG. 14) or with 50 nM Erk2 plus 2.5 nM Mek1 S218D
S222E (labelled lanes 5 to 8 in FIG. 14) in the presence of 1.0 mM
ATP, 50 mM Hepes/KOH, 5 mM magnesium chloride and 0.5 mM DTT at
30.degree. C. At the indicated time points (0, 10, 20 and 40
minutes, see FIG. 14), samples were taken from the reaction,
diluted in SDS sample buffer containing 50 mM EDTA and separated by
SDS polyacrylamide gel electrophoresis (SDS-PAGE). SDS-PAGE
separated reaction samples were blotted onto nitrocellulose and
probed with an antibody against a phospho-eptipe, essential for the
activation of Mnk (anti-Phospho-Mnk Thr197/202; Cell Signaling
Technology, Inc., Beverly, Mass.). The anti-Phospho-Mnk antibody
was detected with a peroxidase-coupled anti rabbit antibody
(Sigma-Aldrich, St. Louis, Mo.) as described elsewhere (Harlow and
Lane, 1998, Antibodies, Cold Spring Harbor Laboratory Press,
NY).
[0223] As the reaction progresses, the activation of Mnk2a can be
visualized by Mnks immuno-reactivity with the anti Phospho-Mnk
antibody (see upper panel in FIG. 14). In addition, Mnk2a was
visualized by Coomassie staining of the gel. Arrows indicate the
Coomassie stained Mhk2a as its mobility is retarded with increasing
phosphorylation (see lower panel in FIG. 14).
[0224] The generation of the phospho-epitope, essential for the
activation of Mnk2a, and the high degree of efficiency of this
process (as shown by the nearly complete electrophoretic mobility
shift) demonstrate the suitablility of this approach to produce
enzymatically active Mnk2a.
[0225] For the validation of the assay, known Mnk inhibitors such
as CGP57380 or CGP025088 may be used (see, Knauf et al., 2001, Mol.
Cell. Biol. 21:5500, Tschopp et al., 2000, Mol Cell Biol Res Comm
3:205 and Slentz-Kesler et al., 2000, Genomics 69:63). As a
negative control, CGP052088 may be used.
[0226] Alternatively, the screening may comprise the use of
cellular based screening systems, e.g. prokaryotic or eukaryotic
cells which overexpress Mnk proteins. Furthermore, transgenic
animals capable of overexpressing or underexpressing Mnk2 and/or
Mnk1 may be used.
[0227] All publications and patents mentioned in the above
specification are herein incorporated by reference.
[0228] Various modifications and variations of the described method
and system of the invention will be apparent to those skilled in
the art without departing from the scope and spirit of the
invention. Although the invention has been described in connection
with specific preferred embodiments, it should be understood that
the invention as claimed should not be unduly limited to such
specific embodiments. Indeed, various modifications of the
described modes for carrying out the invention which are obvious to
those skilled in molecular biology or related fields are intended
to be within the scope of the following claims.
Sequence CWU 1
1
53 1 1444 DNA Homo sapiens 1 cggtcccctc ccccgctggc ggggcccgga
cagaagatgg tgcagaagaa accagccgaa 60 cttcagggtt tccaccgttc
gttcaagggg cagaacccct tcgagctggc cttctcccta 120 gaccagcccg
accacggaga ctctgacttt ggcctgcagt gctcagcccg ccctgacatg 180
cccgccagcc agcccattga catcccggac gccaagaaga ggggcaagaa gaagaagcgc
240 ggccgggcca ccgacagctt ctcgggcagg tttgaagacg tctaccagct
gcaggaagat 300 gtgctggggg agggcgctca tgcccgagtg cagacctgca
tcaacctgat caccagccag 360 gagtacgccg tcaagatcat tgagaagcag
ccaggccaca ttcggagcag ggttttcagg 420 gaggtggaga tgctgtacca
gtgccaggga cacaggaacg tcctagagct gattgagttc 480 ttcgaggagg
aggaccgctt ctacctggtg tttgagaaga tgcggggagg ctccatcctg 540
agccacatcc acaagcgccg gcacttcaac gagctggagg ccagcgtggt ggtgcaggac
600 gtggccagcg ccttggactt tctgcataac aaaggcatcg cccacaggga
cctaaagccg 660 gaaaacatcc tctgtgagca ccccaaccag gtctcccccg
tgaagatctg tgacttcgac 720 ctgggcagcg gcatcaaact caacggggac
tgctccccta tctccacccc ggagctgctc 780 actccgtgcg gctcggcgga
gtacatggcc ccggaggtag tggaggcctt cagcgaggag 840 gctagcatct
acgacaagcg ctgcgacctg tggagcctgg gcgtcatctt gtatatccta 900
ctcagcggct acccgccctt cgtgggccgc tgtggcagcg actgcggctg ggaccgcggc
960 gaggcctgcc ctgcctgcca gaacatgctg tttgagagca tccaggaggg
caagtacgag 1020 ttccccgaca aggactgggc ccacatctcc tgcgctgcca
aagacctcat ctccaagctg 1080 ctggtccgtg acgccaagca gaggctgagt
gccgcccaag tcctgcagca cccctgggtt 1140 caggggtgcg ccccggagaa
caccttgccc actcccatgg tcctgcagag gaacagctgt 1200 gccaaagacc
tcacgtcctt cgcggctgag gccattgcca tgaaccggca gctggcccag 1260
cacgacgagg acctggctga ggaggaggcc gcggggcagg gccagcccgt cctggtccga
1320 gctacctcac gctgcctgca gctgtctcca ccctcccagt ccaagctggc
gcagcggcgg 1380 caaagggcca gtctgtcctc ggccccagtg gtcctggtgg
gagaccacgc ctgaccctcc 1440 catc 1444 2 465 PRT Homo sapiens 2 Met
Val Gln Lys Lys Pro Ala Glu Leu Gln Gly Phe His Arg Ser Phe 1 5 10
15 Lys Gly Gln Asn Pro Phe Glu Leu Ala Phe Ser Leu Asp Gln Pro Asp
20 25 30 His Gly Asp Ser Asp Phe Gly Leu Gln Cys Ser Ala Arg Pro
Asp Met 35 40 45 Pro Ala Ser Gln Pro Ile Asp Ile Pro Asp Ala Lys
Lys Arg Gly Lys 50 55 60 Lys Lys Lys Arg Gly Arg Ala Thr Asp Ser
Phe Ser Gly Arg Phe Glu 65 70 75 80 Asp Val Tyr Gln Leu Gln Glu Asp
Val Leu Gly Glu Gly Ala His Ala 85 90 95 Arg Val Gln Thr Cys Ile
Asn Leu Ile Thr Ser Gln Glu Tyr Ala Val 100 105 110 Lys Ile Ile Glu
Lys Gln Pro Gly His Ile Arg Ser Arg Val Phe Arg 115 120 125 Glu Val
Glu Met Leu Tyr Gln Cys Gln Gly His Arg Asn Val Leu Glu 130 135 140
Leu Ile Glu Phe Phe Glu Glu Glu Asp Arg Phe Tyr Leu Val Phe Glu 145
150 155 160 Lys Met Arg Gly Gly Ser Ile Leu Ser His Ile His Lys Arg
Arg His 165 170 175 Phe Asn Glu Leu Glu Ala Ser Val Val Val Gln Asp
Val Ala Ser Ala 180 185 190 Leu Asp Phe Leu His Asn Lys Gly Ile Ala
His Arg Asp Leu Lys Pro 195 200 205 Glu Asn Ile Leu Cys Glu His Pro
Asn Gln Val Ser Pro Val Lys Ile 210 215 220 Cys Asp Phe Asp Leu Gly
Ser Gly Ile Lys Leu Asn Gly Asp Cys Ser 225 230 235 240 Pro Ile Ser
Thr Pro Glu Leu Leu Thr Pro Cys Gly Ser Ala Glu Tyr 245 250 255 Met
Ala Pro Glu Val Val Glu Ala Phe Ser Glu Glu Ala Ser Ile Tyr 260 265
270 Asp Lys Arg Cys Asp Leu Trp Ser Leu Gly Val Ile Leu Tyr Ile Leu
275 280 285 Leu Ser Gly Tyr Pro Pro Phe Val Gly Arg Cys Gly Ser Asp
Cys Gly 290 295 300 Trp Asp Arg Gly Glu Ala Cys Pro Ala Cys Gln Asn
Met Leu Phe Glu 305 310 315 320 Ser Ile Gln Glu Gly Lys Tyr Glu Phe
Pro Asp Lys Asp Trp Ala His 325 330 335 Ile Ser Cys Ala Ala Lys Asp
Leu Ile Ser Lys Leu Leu Val Arg Asp 340 345 350 Ala Lys Gln Arg Leu
Ser Ala Ala Gln Val Leu Gln His Pro Trp Val 355 360 365 Gln Gly Cys
Ala Pro Glu Asn Thr Leu Pro Thr Pro Met Val Leu Gln 370 375 380 Arg
Asn Ser Cys Ala Lys Asp Leu Thr Ser Phe Ala Ala Glu Ala Ile 385 390
395 400 Ala Met Asn Arg Gln Leu Ala Gln His Asp Glu Asp Leu Ala Glu
Glu 405 410 415 Glu Ala Ala Gly Gln Gly Gln Pro Val Leu Val Arg Ala
Thr Ser Arg 420 425 430 Cys Leu Gln Leu Ser Pro Pro Ser Gln Ser Lys
Leu Ala Gln Arg Arg 435 440 445 Gln Arg Ala Ser Leu Ser Ser Ala Pro
Val Val Leu Val Gly Asp His 450 455 460 Ala 465 3 1549 DNA Homo
sapiens 3 gctggcgggg cccggacaga agatggtgca gaagaaacca gccgaacttc
agggtttcca 60 ccgttcgttc aaggggcaga accccttcga gctggccttc
tccctagacc agcccgacca 120 cggagactct gactttggcc tgcagtgctc
agcccgccct gacatgcccg ccagccagcc 180 cattgacatc ccggacgcca
agaagagggg caagaagaag aagcgcggcc gggccaccga 240 cagcttctcg
ggcaggtttg aagacgtcta ccagctgcag gaagatgtgc tgggggaggg 300
cgctcatgcc cgagtgcaga cctgcatcaa cctgatcacc agccaggagt acgccgtcaa
360 gatcattgag aagcagccag gccacattcg gagcagggtt ttcagggagg
tggagatgct 420 gtaccagtgc cagggacaca ggaacgtcct agagctgatt
gagttcttcg aggaggagga 480 ccgcttctac ctggtgtttg agaagatgcg
gggaggctcc atcctgagcc acatccacaa 540 gcgccggcac ttcaacgagc
tggaggccag cgtggtggtg caggacgtgg ccagcgcctt 600 ggactttctg
cataacaaag gcatcgccca cagggaccta aagccggaaa acatcctctg 660
tgagcacccc aaccaggtct cccccgtgaa gatctgtgac ttcgacctgg gcagcggcat
720 caaactcaac ggggactgct cccctatctc caccccggag ctgctcactc
cgtgcggctc 780 ggcggagtac atggccccgg agttagtgga ggccttcagc
gaggaggcta gcatctacga 840 caagcgctgc gacctgtgga gcctgggcgt
catcttgtat atcctactca gcggctaccc 900 gcccttcgtg ggccgctgtg
gcagcgactg cggctgggac cgcggcgagg cctgccctgc 960 ctgccagaac
atgctgtttg agagcatcca ggagggcaag tacgagttcc ccgacaagga 1020
ctgggcccac atctcctgcg ctgccaaaga cctcatctcc aagctgctgg tccgtgacgc
1080 caagcagagg ctgagtgccg cccaagtcct gcaacacccc tgggttcagg
ggtgcgcccc 1140 ggagaacacc ttgcccactc ccatggtcct gcagaggtgg
gacagtcact tcctcctccc 1200 tccccacccc tgtcgcatcc acgtgcgacc
tggaggactg gtcagaaccg ttactgtgaa 1260 tgagtgaaga tcctggagga
ccctggcccc aggccagctc ccatcgctgg gggacggtga 1320 acggccatgt
gttaatgtta cgatgttttt aaaagacaaa aaaaaaaaaa aaacctcaaa 1380
agttttttta aagtggggga aaaacatcca agcactttaa ttccaatgta ccaggtgaac
1440 tgacggagct cagaagtttt cctttacacc aactgtcaat gccggaattt
tgtattctgt 1500 tttgtaaaga tttaataaaa gtcaaaaaac ttgcaaaaaa
aaaaaaaaa 1549 4 414 PRT Homo sapiens 4 Met Val Gln Lys Lys Pro Ala
Glu Leu Gln Gly Phe His Arg Ser Phe 1 5 10 15 Lys Gly Gln Asn Pro
Phe Glu Leu Ala Phe Ser Leu Asp Gln Pro Asp 20 25 30 His Gly Asp
Ser Asp Phe Gly Leu Gln Cys Ser Ala Arg Pro Asp Met 35 40 45 Pro
Ala Ser Gln Pro Ile Asp Ile Pro Asp Ala Lys Lys Arg Gly Lys 50 55
60 Lys Lys Lys Arg Gly Arg Ala Thr Asp Ser Phe Ser Gly Arg Phe Glu
65 70 75 80 Asp Val Tyr Gln Leu Gln Glu Asp Val Leu Gly Glu Gly Ala
His Ala 85 90 95 Arg Val Gln Thr Cys Ile Asn Leu Ile Thr Ser Gln
Glu Tyr Ala Val 100 105 110 Lys Ile Ile Glu Lys Gln Pro Gly His Ile
Arg Ser Arg Val Phe Arg 115 120 125 Glu Val Glu Met Leu Tyr Gln Cys
Gln Gly His Arg Asn Val Leu Glu 130 135 140 Leu Ile Glu Phe Phe Glu
Glu Glu Asp Arg Phe Tyr Leu Val Phe Glu 145 150 155 160 Lys Met Arg
Gly Gly Ser Ile Leu Ser His Ile His Lys Arg Arg His 165 170 175 Phe
Asn Glu Leu Glu Ala Ser Val Val Val Gln Asp Val Ala Ser Ala 180 185
190 Leu Asp Phe Leu His Asn Lys Gly Ile Ala His Arg Asp Leu Lys Pro
195 200 205 Glu Asn Ile Leu Cys Glu His Pro Asn Gln Val Ser Pro Val
Lys Ile 210 215 220 Cys Asp Phe Asp Leu Gly Ser Gly Ile Lys Leu Asn
Gly Asp Cys Ser 225 230 235 240 Pro Ile Ser Thr Pro Glu Leu Leu Thr
Pro Cys Gly Ser Ala Glu Tyr 245 250 255 Met Ala Pro Glu Val Val Glu
Ala Phe Ser Glu Glu Ala Ser Ile Tyr 260 265 270 Asp Lys Arg Cys Asp
Leu Trp Ser Leu Gly Val Ile Leu Tyr Ile Leu 275 280 285 Leu Ser Gly
Tyr Pro Pro Phe Val Gly Arg Cys Gly Ser Asp Cys Gly 290 295 300 Trp
Asp Arg Gly Glu Ala Cys Pro Ala Cys Gln Asn Met Leu Phe Glu 305 310
315 320 Ser Ile Gln Glu Gly Lys Tyr Glu Phe Pro Asp Lys Asp Trp Ala
His 325 330 335 Ile Ser Cys Ala Ala Lys Asp Leu Ile Ser Lys Leu Leu
Val Arg Asp 340 345 350 Ala Lys Gln Arg Leu Ser Ala Ala Gln Val Leu
Gln His Pro Trp Val 355 360 365 Gln Gly Cys Ala Pro Glu Asn Thr Leu
Pro Thr Pro Met Val Leu Gln 370 375 380 Arg Trp Asp Ser His Phe Leu
Leu Pro Pro His Pro Cys Arg Ile His 385 390 395 400 Val Arg Pro Gly
Gly Leu Val Arg Thr Val Thr Val Asn Glu 405 410 5 2745 DNA Homo
sapiens 5 ggcacgaggg cgaccgctcc ccggcgggag ccagcgaagg tttccatgtc
agaggccgat 60 ggagaactga agattgccac ctacgcacaa aggccattga
gacacttcgt gtagctggaa 120 gacaccaact tcctgacagg agctttattt
catttgggat ttcaagttta cagatggtat 180 cttctcaaaa gttggaaaaa
cctatagaga tgggcagtag cgaacccctt cccatcgcag 240 atggtgacag
gaggaggaag aagaagcgga ggggccgggc cactgactcc ttgccaggaa 300
agtttgaaga tatgtacaag ctgacctctg aattgcttgg agagggagcc tatgccaaag
360 ttcaaggtgc cgtgagccta cagaatggca aagagtatgc cgtcaaaatc
atcgagaaac 420 aagcagggca cagtcggagt agggtgtttc gagaggtgga
gacgctgtat cagtgtcagg 480 gaaacaagaa cattttggag ctgattgagt
tctttgaaga tgacacaagg ttttacttgg 540 tctttgagaa attgcaagga
ggttccatct tagcccacat ccagaagcaa aagcacttca 600 atgagcgaga
agccagccga gtggtgcggg acgttgctgc tgcccttgac ttcctgcata 660
ccaaagacaa agtctctctc tgtcacctag gctggagtgc tatggcgcca tcagggctca
720 ctgcagcccc aacctccctg ggctccagtg atcctcccac ctcagcctcc
caagtagctg 780 ggactacagg cattgctcat cgtgatctga aaccagaaaa
tatattgtgt gaatctccag 840 aaaaggtgtc tccagtgaaa atctgtgact
ttgacttggg cagtgggatg aaactgaaca 900 actcctgtac ccccataacc
acaccagagc tgaccacccc atgtggctct gcagaataca 960 tggcccctga
ggtagtggag gtcttcacgg accaggccac attctacgac aagcgctgtg 1020
acctgtggag cctgggcgtg gtcctctaca tcatgctgag tggctaccca cccttcgtgg
1080 gtcactgcgg ggccgactgt ggctgggacc ggggcgaggt ctgcagggtg
tgccagaaca 1140 agctgtttga aagcatccag gaaggcaagt atgagtttcc
tgacaaggac tgggcacaca 1200 tctccagtga agccaaagac ctcatctcca
agctcctggt gcgagatgca aagcagagac 1260 ttagcgccgc ccaagttctg
cagcacccat gggtgcaggg gcaagctcca gaaaagggac 1320 tccccacgcc
gcaagtcctc cagaggaaca gcagcacaat ggacctgacg ctcttcgcag 1380
ctgaggccat cgcccttaac cgccagctat ctcagcacga agagaacgaa ctagcagagg
1440 agccagaggc actagctgat ggcctctgct ccatgaagct ttcccctccc
tgcaagtcac 1500 gcctggcccg gagacgggcc ctggcccagg caggccgtgg
tgaagacagg agcccgccca 1560 cagcactctg aaatgctcca gtcacacctt
ataggcccta ggcctggcca ggcattgtcc 1620 cctggaaacc tgtgtggcta
aagtctgctg agcaggcagc agcctctgct ctgtggctcc 1680 attcaggctt
tttcatctac gaaggccctg aggttcccat caacccccat ttccctaggg 1740
tcctggagga aaaagctttt tccaaagggg ttgtctttga aaaggaaagc aatcacttct
1800 cactttgcat aattgcctgc agcaggaaca tctcttcact gggctccacc
tgctcacccg 1860 cctgcagatc tgggatccag cctgctctca ccgctgtagc
tgtggcggct ggggctgcag 1920 cctgcaggga gaagcaagaa gcatcagttg
acagaggctg ccgacacgtg cctcttccct 1980 ctcttctctg tcaccctcct
ctggcggtcc ttccaccttc ctctgtcctc cggatgtcct 2040 ctttgcccgt
cttctccctt ggctgagcaa agccatcccc tcaattcagg gaagggcaag 2100
gagccttcct cattcaggaa atcaaatcag tcttccggtc tgcagcacgg aaaagcacat
2160 aatctttctt tgctgtgact gaaatgtatc cctcgtttat catccccttt
gtttgtgatt 2220 gctgctaaag tcagtagtat cgttttttta aaaaaaaagt
ttggtgtttt taaccatgct 2280 gttccagcaa agatgatacc ttaaactccc
actgcaagcc catgaacttc ccagagagtg 2340 gaacggcttg ctcttctttc
tagaatgtcc atgcacttgg gttttaatca gcagttccct 2400 attattctga
ttttaagctg ttcctgtgat gaacttagag acagcatcgg tgtctgctgc 2460
tgtgtcccca ggtcttgtgt gggtggcaca gatctgggca gttagatagt gctctgtgcc
2520 taaggtgaag ccacactagg gtgaagcctc acttccctgt ttgagcaatg
cagtgcctgc 2580 tgcccgtgtg catgaaggta cagccattca gataagtgga
actattgagt tacataaaga 2640 aaatagattt gcatttgtca ggcagacgtt
tatacaacac cacggtgctt ttatacattg 2700 tgcttatttt aataaaactg
aaattctaaa aaaaaaaaaa aaaaa 2745 6 465 PRT Homo sapiens 6 Met Val
Ser Ser Gln Lys Leu Glu Lys Pro Ile Glu Met Gly Ser Ser 1 5 10 15
Glu Pro Leu Pro Ile Ala Asp Gly Asp Arg Arg Arg Lys Lys Lys Arg 20
25 30 Arg Gly Arg Ala Thr Asp Ser Leu Pro Gly Lys Phe Glu Asp Met
Tyr 35 40 45 Lys Leu Thr Ser Glu Leu Leu Gly Glu Gly Ala Tyr Ala
Lys Val Gln 50 55 60 Gly Ala Val Ser Leu Gln Asn Gly Lys Glu Tyr
Ala Val Lys Ile Ile 65 70 75 80 Glu Lys Gln Ala Gly His Ser Arg Ser
Arg Val Phe Arg Glu Val Glu 85 90 95 Thr Leu Tyr Gln Cys Gln Gly
Asn Lys Asn Ile Leu Glu Leu Ile Glu 100 105 110 Phe Phe Glu Asp Asp
Thr Arg Phe Tyr Leu Val Phe Glu Lys Leu Gln 115 120 125 Gly Gly Ser
Ile Leu Ala His Ile Gln Lys Gln Lys His Phe Asn Glu 130 135 140 Arg
Glu Ala Ser Arg Val Val Arg Asp Val Ala Ala Ala Leu Asp Phe 145 150
155 160 Leu His Thr Lys Asp Lys Val Ser Leu Cys His Leu Gly Trp Ser
Ala 165 170 175 Met Ala Pro Ser Gly Leu Thr Ala Ala Pro Thr Ser Leu
Gly Ser Ser 180 185 190 Asp Pro Pro Thr Ser Ala Ser Gln Val Ala Gly
Thr Thr Gly Ile Ala 195 200 205 His Arg Asp Leu Lys Pro Glu Asn Ile
Leu Cys Glu Ser Pro Glu Lys 210 215 220 Val Ser Pro Val Lys Ile Cys
Asp Phe Asp Leu Gly Ser Gly Met Lys 225 230 235 240 Leu Asn Asn Ser
Cys Thr Pro Ile Thr Thr Pro Glu Leu Thr Thr Pro 245 250 255 Cys Gly
Ser Ala Glu Tyr Met Ala Pro Glu Val Val Glu Val Phe Thr 260 265 270
Asp Gln Ala Thr Phe Tyr Asp Lys Arg Cys Asp Leu Trp Ser Leu Gly 275
280 285 Val Val Leu Tyr Ile Met Leu Ser Gly Tyr Pro Pro Phe Val Gly
His 290 295 300 Cys Gly Ala Asp Cys Gly Trp Asp Arg Gly Glu Val Cys
Arg Val Cys 305 310 315 320 Gln Asn Lys Leu Phe Glu Ser Ile Gln Glu
Gly Lys Tyr Glu Phe Pro 325 330 335 Asp Lys Asp Trp Ala His Ile Ser
Ser Glu Ala Lys Asp Leu Ile Ser 340 345 350 Lys Leu Leu Val Arg Asp
Ala Lys Gln Arg Leu Ser Ala Ala Gln Val 355 360 365 Leu Gln His Pro
Trp Val Gln Gly Gln Ala Pro Glu Lys Gly Leu Pro 370 375 380 Thr Pro
Gln Val Leu Gln Arg Asn Ser Ser Thr Met Asp Leu Thr Leu 385 390 395
400 Phe Ala Ala Glu Ala Ile Ala Leu Asn Arg Gln Leu Ser Gln His Glu
405 410 415 Glu Asn Glu Leu Ala Glu Glu Pro Glu Ala Leu Ala Asp Gly
Leu Cys 420 425 430 Ser Met Lys Leu Ser Pro Pro Cys Lys Ser Arg Leu
Ala Arg Arg Arg 435 440 445 Ala Leu Ala Gln Ala Gly Arg Gly Glu Asp
Arg Ser Pro Pro Thr Ala 450 455 460 Leu 465 7 1253 DNA Drosophila
melanogaster misc_feature (5)..(5) n is a, c, g, or t 7 cgagnaagtg
ttactatcta aacacatttc aaacaattct taacaaacaa ttccaaacat 60
acaattccac ttaccactta ccgaccaaat tacgagttta caatggacaa agctgaacgc
120 gactactggc atcttcgatc cttggaaatc gaagaggagc cgcgatttcc
gccaacaaac 180 gtcgctgatc cactaaccgc acgcaatctg ttccagctct
acgtcaacac cttcattgga 240 gccaatctgg ccgagtcgtg tgttttccca
ttggacgtgg ccaagacccg gatgcaggta 300 gatggcgagc aggccaagaa
gacgggtaaa gcgatgccaa ctttccgtgc aactcttacc 360 aacatgatcc
gagtggaggg attcaagtcg ctctacgccg gcttctcggc aatggtgacc 420
cgaaacttta tcttcaactc gttacgtgtt gttctctacg acgttttccg gcgccctttt
480 ctctaccaga acgaacggaa cgaggaagtg ctcaagatct acatggcgct
gggatgcagc 540 ttcaccgcag gctgcattgc ccaggcactg gccaatccct
ttgacatcgt caaggtgcga 600 atgcagacgg aaggacgccg ccgccagctg
ggctatgatg tgcgggtgaa cagcatggtg 660 caggccttcg tggacatcta
ccgccgtggc ggactgccca gtatgtggaa gggtgtaggg 720 cccagctgca
tgcgtgcctg cctgatgacg accggcgatg tgggcagtta cgatatcagt 780
aagcgcacct tcaagcgcct gctggacttg
gaggaaggcc tgccactgcg tttcgtgtct 840 tccatgtgcg ccggactaac
ggcatccgtg ctcagcacgc cggcgaacgt gatcaagtcg 900 cggatgatga
accagccggt gaacgagagc ggcaagaatc tgtactacaa gaactccctc 960
gactgcatta ggaagctggt cagggaggag ggtgtcctca cgttgtataa gggcctcatg
1020 cccacttggt ttcgcctggg accgttctca gtgctctttt ggctgtccgt
cgagcagctg 1080 cgtcagtgga aaggccagag tggattttag gagcaaacta
tcaatcttac tatcgtattt 1140 tgtatgtctt ttaacacgca ataaaaaggg
tgcaagtcaa accatctatt atacatatta 1200 taaatataac tttaatccca
aaaaaaaaaa aaaaaactcg tgccgaattc gat 1253 8 335 PRT Drosophila
melanogaster 8 Met Asp Lys Ala Glu Arg Asp Tyr Trp His Leu Arg Ser
Leu Glu Ile 1 5 10 15 Glu Glu Glu Pro Arg Phe Pro Pro Thr Asn Val
Ala Asp Pro Leu Thr 20 25 30 Ala Arg Asn Leu Phe Gln Leu Tyr Val
Asn Thr Phe Ile Gly Ala Asn 35 40 45 Leu Ala Glu Ser Cys Val Phe
Pro Leu Asp Val Ala Lys Thr Arg Met 50 55 60 Gln Val Asp Gly Glu
Gln Ala Lys Lys Thr Gly Lys Ala Met Pro Thr 65 70 75 80 Phe Arg Ala
Thr Leu Thr Asn Met Ile Arg Val Glu Gly Phe Lys Ser 85 90 95 Leu
Tyr Ala Gly Phe Ser Ala Met Val Thr Arg Asn Phe Ile Phe Asn 100 105
110 Ser Leu Arg Val Val Leu Tyr Asp Val Phe Arg Arg Pro Phe Leu Tyr
115 120 125 Gln Asn Glu Arg Asn Glu Glu Val Leu Lys Ile Tyr Met Ala
Leu Gly 130 135 140 Cys Ser Phe Thr Ala Gly Cys Ile Ala Gln Ala Leu
Ala Asn Pro Phe 145 150 155 160 Asp Ile Val Lys Val Arg Met Gln Thr
Glu Gly Arg Arg Arg Gln Leu 165 170 175 Gly Tyr Asp Val Arg Val Asn
Ser Met Val Gln Ala Phe Val Asp Ile 180 185 190 Tyr Arg Arg Gly Gly
Leu Pro Ser Met Trp Lys Gly Val Gly Pro Ser 195 200 205 Cys Met Arg
Ala Cys Leu Met Thr Thr Gly Asp Val Gly Ser Tyr Asp 210 215 220 Ile
Ser Lys Arg Thr Phe Lys Arg Leu Leu Asp Leu Glu Glu Gly Leu 225 230
235 240 Pro Leu Arg Phe Val Ser Ser Met Cys Ala Gly Leu Thr Ala Ser
Val 245 250 255 Leu Ser Thr Pro Ala Asn Val Ile Lys Ser Arg Met Met
Asn Gln Pro 260 265 270 Val Asn Glu Ser Gly Lys Asn Leu Tyr Tyr Lys
Asn Ser Leu Asp Cys 275 280 285 Ile Arg Lys Leu Val Arg Glu Glu Gly
Val Leu Thr Leu Tyr Lys Gly 290 295 300 Leu Met Pro Thr Trp Phe Arg
Leu Gly Pro Phe Ser Val Leu Phe Trp 305 310 315 320 Leu Ser Val Glu
Gln Leu Arg Gln Trp Lys Gly Gln Ser Gly Phe 325 330 335 9 24 DNA
Drosophila melanogaster 9 ctaaacaaac aattccaaac atag 24 10 24 DNA
Drosophila melanogaster 10 aaaagacata gaaaatacga tagt 24 11 18 DNA
Mus musculus 11 gctgagggcc tctgctcc 18 12 16 DNA Mus musculus 12
tcgccttcga gccagg 16 13 27 DNA Mus musculus misc_feature (1)..(1)
FAM reporter dye 13 tgaagctgtc ccctccatcc aaatctc 27 14 19 DNA Mus
musculus 14 tgcacttgat tgaccccga 19 15 22 DNA Mus musculus 15
tttctgattg tcaaccctcc aa 22 16 24 DNA Mus musculus misc_feature
(1)..(1) FAM reporter dye 16 ccccatcatc cacctgcagt gtcc 24 17 23
DNA Homo sapiens 17 ccatctcccc ctctgtacat agg 23 18 19 DNA Homo
sapiens 18 ccggctggcg atagcttaa 19 19 27 DNA Homo sapiens
misc_feature (1)..(1) FAM reporter dye 19 cacccgtccc ccaatcaaat
ctaaagg 27 20 26 DNA Homo sapiens 20 ttactgtgaa tgagtgaaga tcctgg
26 21 17 DNA Homo sapiens 21 atggccgttc accgtcc 17 22 21 DNA Homo
sapiens misc_feature (1)..(1) FAM reporter dye 22 ccaggccagc
tcccatcgct g 21 23 21 DNA Mus musculus 23 aagttggcct tcgcgttaga c
21 24 20 DNA Mus musculus 24 cgatatgtac aaggagctag 20 25 44 DNA Mus
musculus 25 ctcccccatc tccgcaccag agctgctcgc cccgtgtggg tcag 44 26
41 DNA Mus musculus 26 ctgacccaca cggggcgagc tctggtgcgg agatggggga
g 41 27 19 DNA Mus musculus 27 gctgctggtc cgagatgcc 19 28 18 DNA
Mus musculus 28 gggtcatgcg cgatcccc 18 29 22 DNA Mus musculus 29
cagcgtggta gtacaggacg tg 22 30 16 DNA Mus musculus 30 tccctgtggg
cgatgc 16 31 28 DNA Mus musculus 31 cagtgccctg gacttccctg cataacaa
28 32 19 DNA Mus musculus 32 acatcagccc acagtgtga 19 33 21 DNA Mus
musculus 33 tctccattga gtttgatacc a 21 34 27 DNA Mus musculus
misc_feature (1)..(6) non priming nucleotides/ attached EcoR1
restriction site 34 cggaatccac tagctccttg tacatat 27 35 29 DNA Mus
musculus misc_feature (1)..(8) non priming nucleotides/ attached
Cla1 restriction site 35 ccatcgatgg aactcgtatt gcatagtag 29 36 23
DNA Mus musculus 36 agactaggga ggagggtgga gga 23 37 22 DNA Mus
musculus 37 ggtggatgtg gaatgtgtgc ga 22 38 21 DNA Mus musculus 38
ggggtgtagg ggtctgttag g 21 39 424 PRT Homo sapiens 39 Met Val Ser
Ser Gln Lys Leu Glu Lys Pro Ile Glu Met Gly Ser Ser 1 5 10 15 Glu
Pro Leu Pro Ile Ala Asp Gly Asp Arg Arg Arg Lys Lys Lys Arg 20 25
30 Arg Gly Arg Ala Thr Asp Ser Leu Pro Gly Lys Phe Glu Asp Met Tyr
35 40 45 Lys Leu Thr Ser Glu Leu Leu Gly Glu Gly Ala Tyr Ala Lys
Val Gln 50 55 60 Gly Ala Val Ser Leu Gln Asn Gly Lys Glu Tyr Ala
Val Lys Ile Ile 65 70 75 80 Glu Lys Gln Ala Gly His Ser Arg Ser Arg
Val Phe Arg Glu Val Glu 85 90 95 Thr Leu Tyr Gln Cys Gln Gly Asn
Lys Asn Ile Leu Glu Leu Ile Glu 100 105 110 Phe Phe Glu Asp Asp Thr
Arg Phe Tyr Leu Val Phe Glu Lys Leu Gln 115 120 125 Gly Gly Ser Ile
Leu Ala His Ile Gln Lys Gln Lys His Phe Asn Glu 130 135 140 Arg Glu
Ala Ser Arg Val Val Arg Asp Val Ala Ala Ala Leu Asp Phe 145 150 155
160 Leu His Thr Lys Gly Ile Ala His Arg Asp Leu Lys Pro Glu Asn Ile
165 170 175 Leu Cys Glu Ser Pro Glu Lys Val Ser Pro Val Lys Ile Cys
Asp Phe 180 185 190 Asp Leu Gly Ser Gly Met Lys Leu Asn Asn Ser Cys
Thr Pro Ile Thr 195 200 205 Thr Pro Glu Leu Thr Thr Pro Cys Gly Ser
Ala Glu Tyr Met Ala Pro 210 215 220 Glu Val Val Glu Val Phe Thr Asp
Gln Ala Thr Phe Tyr Asp Lys Arg 225 230 235 240 Cys Asp Leu Trp Ser
Leu Gly Val Val Leu Tyr Ile Met Leu Ser Gly 245 250 255 Tyr Pro Pro
Phe Val Gly His Cys Gly Ala Asp Cys Gly Trp Asp Arg 260 265 270 Gly
Glu Val Cys Arg Val Cys Gln Asn Lys Leu Phe Glu Ser Ile Gln 275 280
285 Glu Gly Lys Tyr Glu Phe Pro Asp Lys Asp Trp Ala His Ile Ser Ser
290 295 300 Glu Ala Lys Asp Leu Ile Ser Lys Leu Leu Val Arg Asp Ala
Lys Gln 305 310 315 320 Arg Leu Ser Ala Ala Gln Val Leu Gln His Pro
Trp Val Gln Gly Gln 325 330 335 Ala Pro Glu Lys Gly Leu Pro Thr Pro
Gln Val Leu Gln Arg Asn Ser 340 345 350 Ser Thr Met Asp Leu Thr Leu
Phe Ala Ala Glu Ala Ile Ala Leu Asn 355 360 365 Arg Gln Leu Ser Gln
His Glu Glu Asn Glu Leu Ala Glu Glu Pro Glu 370 375 380 Ala Leu Ala
Asp Gly Leu Cys Ser Met Lys Leu Ser Pro Pro Cys Lys 385 390 395 400
Ser Arg Leu Ala Arg Arg Arg Ala Leu Ala Gln Ala Gly Arg Gly Glu 405
410 415 Asp Arg Ser Pro Pro Thr Ala Leu 420 40 1150 PRT Drosophila
melanogaster 40 Met Val Glu Pro Lys Ser Gly Thr Ala Ala Ser Ala Ala
Ala Ala Lys 1 5 10 15 Ala Ser Asn Asn Asn Asn Asn Asn His Pro Arg
Gly Ser Gly Asp Ser 20 25 30 Gly Ile Arg Ser Gly Ser Gly Ile Ser
Cys Ser Asn Thr Asp Asn Ser 35 40 45 Cys Ser Gln Ser Gln Ser Asp
Gly Gln Asn Glu Leu Thr Arg Tyr Ser 50 55 60 Ser Glu Asp Val Ser
Gly Asn Glu Ser Ser Glu Ala Pro Asn Met Thr 65 70 75 80 Glu Val Glu
Arg Gln Ala Glu Leu Asn Arg His Lys Glu Glu Met Gln 85 90 95 Lys
Lys Arg Arg Lys Lys Arg Ile Ser Ser Ser Leu His Ser Ser Thr 100 105
110 Phe Gln Glu Leu Tyr Lys Leu Thr Gly Glu Ile Leu Gly Glu Gly Ala
115 120 125 Tyr Ala Ser Val Gln Thr Cys Val Asn Ile Tyr Thr Asp Leu
Glu Tyr 130 135 140 Ala Val Lys Val Ile Asp Lys Ile Pro Gly His Ala
Arg Ala Arg Val 145 150 155 160 Phe Arg Glu Val Glu Thr Phe His His
Cys Gln Gly His Leu Gly Ile 165 170 175 Leu Gln Leu Ile Glu Phe Phe
Glu Asp Asp Lys Lys Phe Tyr Leu Val 180 185 190 Phe Glu Lys Ile Asn
Gly Gly Pro Leu Leu Ser Arg Ile Gln Glu His 195 200 205 Ile Cys Phe
Ser Glu His Glu Pro Ser Gln Ile Ile Lys Glu Ile Ala 210 215 220 Ser
Gly Leu Asp Phe Leu His Lys Lys Gly Ile Ala His Arg Asp Leu 225 230
235 240 Lys Pro Glu Asn Ile Leu Cys Val Lys Thr Asp Ser Leu Cys Pro
Ile 245 250 255 Lys Ile Cys Asp Phe Asp Leu Gly Ser Gly Ile Lys Phe
Thr Thr Asp 260 265 270 Ile Ser Ser Pro Ala Ala Thr Pro Gln Leu Leu
Thr Pro Val Gly Ser 275 280 285 Ala Glu Phe Met Ala Pro Glu Val Val
Asp Leu Phe Val Gly Glu Ala 290 295 300 His Tyr Tyr Asp Lys Arg Cys
Asp Leu Trp Ser Leu Gly Val Ile Ala 305 310 315 320 Tyr Ile Leu Leu
Cys Gly Tyr Pro Pro Phe Ser Gly Asn Cys Gly Glu 325 330 335 Asp Cys
Gly Trp Asn Arg Gly Glu Asn Cys Arg Thr Cys Gln Glu Leu 340 345 350
Leu Phe Glu Ser Ile Gln Glu Gly His Phe Ser Phe Pro Glu Ala Glu 355
360 365 Trp His Asp Val Ser Asp Glu Ala Lys Asp Leu Ile Ser Asn Leu
Leu 370 375 380 Val Lys Lys Ala Ser Asn Arg Leu Ser Ala Glu Ala Val
Leu Asn His 385 390 395 400 Pro Trp Ile Arg Met Cys Glu Gln Glu Pro
Pro Ala Ser Lys His Gly 405 410 415 Arg Arg His Lys Ala Leu Gln Thr
Pro Ser Asn Ile Arg Arg Asn His 420 425 430 Gln Ser Ala Arg Glu Ile
Ser Gln Phe Ala Glu Ser Ala Met Ala Val 435 440 445 Lys Arg Val Val
Leu Gln His Phe Ser Met Arg Tyr Asp Tyr Met Lys 450 455 460 Glu Arg
Pro Asn Ile Tyr Gln Pro Ser Gln Ala Tyr Met Asp Ala Tyr 465 470 475
480 Ser Asp Glu Asn Tyr Asn Pro Lys Pro Pro Gly His Tyr Thr Arg Asn
485 490 495 Arg Ser Gln Arg Asn Pro Ala Ser Ser Leu Cys Gly Tyr Gly
Gly Arg 500 505 510 Met Ser Ser Met His Gly Gln Arg Ala Asn Ser Arg
Arg Ser Ser Arg 515 520 525 Asn Ala Ser Arg Asn Ala Ser Ala Ile Tyr
Pro Asn Ser Gly Gly Phe 530 535 540 Lys Thr Leu Asn Val His Glu Glu
Asp Asp Asp Asp Glu Gly Leu Glu 545 550 555 560 Ala Phe Gly His Ile
Asp Asp Asp Asp Glu Trp Ser Arg Ser Arg Arg 565 570 575 Glu Tyr Gln
Gln Gln Cys Glu Thr Leu Gly Glu Asp Arg Phe Arg Arg 580 585 590 Gln
Ser Gly Ser Glu Gly Asp Glu Val Glu Asp Asp Glu Asp Gly Glu 595 600
605 Asn Glu Asp Tyr Gln His Tyr Lys His Tyr Trp Arg Glu Leu Asp Glu
610 615 620 Glu Glu Gly Asp Asp Tyr Leu Tyr Glu Gln Gln Gln Arg Val
Asp Asp 625 630 635 640 Lys Phe Gly Glu Glu Glu Phe Glu Asp Glu Pro
Lys Glu Glu Thr Gln 645 650 655 Ala Asp Asn Leu Lys Leu Ser Lys Ala
Tyr Val Glu Gln Val Gly Glu 660 665 670 Thr Asn Val Glu Lys Ser Lys
Pro Gln Asp Asp Asn Gly Gly Tyr Ile 675 680 685 Arg Glu Asp Leu Ile
Met Asp Asn Met Asp Met Lys Lys Asn Thr Gln 690 695 700 Gln Ser Glu
Phe Ala Lys Leu Thr Ile Met Arg Asn Asp Ala Gln Thr 705 710 715 720
Glu Glu Asn Lys Ile Met Gln Gln Gln Asp Glu Glu Lys Lys Glu Asp 725
730 735 Lys Gln Gln Asp Asp Val Asp Gly Ala Lys Lys Gln Gly Pro Ser
Ser 740 745 750 Asp Ile Ser Ala Thr Thr Ile Thr Asp Asn Asn Lys Leu
Gln Thr Pro 755 760 765 Val Met Thr Thr Thr His Ile Asn Asn Trp Gln
Thr Gly Asp Ala Ile 770 775 780 Glu Asp Asp Asp Val Lys Leu Leu Asp
Ser Ile Ser Asp Leu Asn Glu 785 790 795 800 Lys Leu Pro Glu Ile Tyr
Glu Thr Ala Asn Ile Val Val Asn Ser Ala 805 810 815 Ala Val Pro Ala
Ala Ser Thr Pro Ala Ala Ser Ala Thr Arg Pro Pro 820 825 830 Thr Asp
Asn Pro Glu Glu Asp Asp Ser Asn Val Thr Lys Pro Thr Thr 835 840 845
Thr Ala Glu Gly Thr Thr Met Gln Thr Thr Phe Gly Met Ser Ala Glu 850
855 860 Glu Glu Lys Pro Val Ala Leu Ser His Thr Ala Gly His His Ser
Lys 865 870 875 880 Thr Gly Arg Thr Val Asn Phe Ala Pro Asp Ala Tyr
Gln Asn Asp Glu 885 890 895 Asp Ala Asp Ile Asp Glu Asp Asp Asp Tyr
Asp Asp Glu Glu Asn Leu 900 905 910 His Glu His Ser Lys Gln Gln Leu
Pro Ser Asn Ala Tyr Thr Arg Lys 915 920 925 Gln Arg Gln Gln His Gln
Arg Tyr Ile Val Pro Arg Tyr Gln Leu Ala 930 935 940 Asp Gln Val Pro
Gln Arg Gln His Thr Glu Asn Trp Arg Tyr Arg Thr 945 950 955 960 His
His Ser Gln Glu Gln Gln Pro Thr Ala Asp Tyr Arg Lys Tyr Arg 965 970
975 Pro Pro Phe Ser Thr Gly Gly Gly Gly Gly His His Gly Asn Leu Gln
980 985 990 Arg Asn Tyr Leu Gly Ser Phe Ser His Ser Gly Gly Ala Ala
Gly Tyr 995 1000 1005 Lys Ile Ala Pro Met Pro Pro Pro Met Gln Pro
Pro Pro Arg His 1010 1015 1020 Asn Ser Ala Gly Ser Ser Gly Ser Gly
Ser Gly Ser Gly Gly Ser 1025 1030 1035 Pro Pro Ser Asp Glu Gln Ser
Ala Ile Arg Asn Trp Arg Gln Asp 1040 1045 1050 Cys Val Tyr Ala Arg
Ser Cys Gly Met Asn Gln Gly Pro Glu Gln 1055 1060 1065 Gln Arg His
Asn Arg Ser Ser Gly Gln Arg Val Gln Gln Gln Pro 1070 1075 1080 Arg
Ile Gly Ser Gly Arg Phe Ala His Leu Gln Ala Ala Gln Leu 1085 1090
1095 Met Asp Glu Leu Pro Asp Met Arg Ile Gly Leu Ser Pro Pro Ser
1100 1105 1110 Glu Ser Val Leu Leu Gln Arg Arg Leu Arg Gln Gln Gln
Arg Ala 1115 1120 1125 Asn Asp Leu Ser Glu Val Leu Arg Ala Gly His
Arg Gln Trp Leu 1130 1135 1140 Arg Gly Ser Thr
Val Asp Arg 1145 1150 41 414 PRT Homo sapiens 41 Met Val Gln Lys
Lys Pro Ala Glu Leu Gln Gly Phe His Arg Ser Phe 1 5 10 15 Lys Gly
Gln Asn Pro Phe Glu Leu Ala Phe Ser Leu Asp Gln Pro Asp 20 25 30
His Gly Asp Ser Asp Phe Gly Leu Gln Cys Ser Ala Arg Pro Asp Met 35
40 45 Pro Ala Ser Gln Pro Ile Asp Ile Pro Asp Ala Lys Lys Arg Gly
Lys 50 55 60 Lys Lys Lys Arg Gly Arg Ala Thr Asp Ser Phe Ser Gly
Arg Phe Glu 65 70 75 80 Asp Val Tyr Gln Leu Gln Glu Asp Val Leu Gly
Glu Gly Ala His Ala 85 90 95 Arg Val Gln Thr Cys Ile Asn Leu Ile
Thr Ser Gln Glu Tyr Ala Val 100 105 110 Lys Ile Ile Glu Lys Gln Pro
Gly His Ile Arg Ser Arg Val Phe Arg 115 120 125 Glu Val Glu Met Leu
Tyr Gln Cys Gln Gly His Arg Asn Val Leu Glu 130 135 140 Leu Ile Glu
Phe Phe Glu Glu Glu Asp Arg Phe Tyr Leu Val Phe Glu 145 150 155 160
Lys Met Arg Gly Gly Ser Ile Leu Ser His Ile His Lys Arg Arg His 165
170 175 Phe Asn Glu Leu Glu Ala Ser Val Val Val Gln Asp Val Ala Ser
Ala 180 185 190 Leu Asp Phe Leu His Asn Lys Gly Ile Ala His Arg Asp
Leu Lys Pro 195 200 205 Glu Asn Ile Leu Cys Glu His Pro Asn Gln Val
Ser Pro Val Lys Ile 210 215 220 Cys Asp Phe Asp Leu Gly Ser Gly Ile
Lys Leu Asn Gly Asp Cys Ser 225 230 235 240 Pro Ile Ser Thr Pro Glu
Leu Leu Thr Pro Cys Gly Ser Ala Glu Tyr 245 250 255 Met Ala Pro Glu
Leu Val Glu Ala Phe Ser Glu Glu Ala Ser Ile Tyr 260 265 270 Asp Lys
Arg Cys Asp Leu Trp Ser Leu Gly Val Ile Leu Tyr Ile Leu 275 280 285
Leu Ser Gly Tyr Pro Pro Phe Val Gly Arg Cys Gly Ser Asp Cys Gly 290
295 300 Trp Asp Arg Gly Glu Ala Cys Pro Ala Cys Gln Asn Met Leu Phe
Glu 305 310 315 320 Ser Ile Gln Glu Gly Lys Tyr Glu Phe Pro Asp Lys
Asp Trp Ala His 325 330 335 Ile Ser Cys Ala Ala Lys Asp Leu Ile Ser
Lys Leu Leu Val Arg Asp 340 345 350 Ala Lys Gln Arg Leu Ser Ala Ala
Gln Val Leu Gln His Pro Trp Val 355 360 365 Gln Gly Cys Ala Pro Glu
Asn Thr Leu Pro Thr Pro Met Val Leu Gln 370 375 380 Arg Trp Asp Ser
His Phe Leu Leu Pro Pro His Pro Cys Arg Ile His 385 390 395 400 Val
Arg Pro Gly Gly Leu Val Arg Thr Val Thr Val Asn Glu 405 410 42 48
DNA Mus musculus 42 ccttagacct cggcccagca cagggaagcc cgacctggtg
agccccca 48 43 48 DNA Mus musculus 43 ctcttctcta cagacatgcc
ttccgcaggt tcgaaggtga gctgcaga 48 44 48 DNA Mus musculus 44
ctcgcattcc agatgtctat cagctatgct gtcaagagct ggggtctg 48 45 48 DNA
Mus musculus 45 cctacattgt agatcattga gaagcaggga cataggtaag
gtggcctg 48 46 49 DNA Mus musculus 46 tcccaactca ggaatgttct
agaagaagat gcgtggcggt aggtactgg 49 47 48 DNA Mus musculus 47
ctggcctgca caggatccat cctagcataa caaaggtgtg gcagggac 48 48 48 DNA
Mus musculus 48 cacctcacta ggcatcgccc acagccccaa ccaggtgagg
ctgcctga 48 49 48 DNA Mus musculus 49 ccattcccag gtctcgccag
tgaagctgct caccccggtg agggcagt 48 50 48 DNA Mus musculus 50
ccctgcccca cagtgtgggt cagctgggtg cagggggtaa gccttggg 48 51 48 DNA
Mus musculus 51 gacttcttac agtgtgcccc agagttggtt ctgcagaggt
gaggcctg 48 52 48 DNA Mus musculus 52 catcctatcc ccaggaacag
ctgtgtcatt taaaaatttc tgtgcagt 48 53 34 DNA Mus musculus 53
aaacacaagc ctaaaaaaaa aaacaagcat gggg 34
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