U.S. patent application number 10/627273 was filed with the patent office on 2004-06-10 for isolated nucleic acid molecules which encode t cell inducible factors (tifs), the proteins encoded, and uses thereof.
Invention is credited to Dumoutier, Laure, Louahed, Jamila, Renauld, Jean-Christophe.
Application Number | 20040110189 10/627273 |
Document ID | / |
Family ID | 27391052 |
Filed Date | 2004-06-10 |
United States Patent
Application |
20040110189 |
Kind Code |
A1 |
Dumoutier, Laure ; et
al. |
June 10, 2004 |
Isolated nucleic acid molecules which encode T cell inducible
factors (TIFs), the proteins encoded, and uses thereof
Abstract
The invention involves isolation of nucleic acid molecules, the
expression of which are upregulated by interleukin-9. The amino
acid sequences of the proteins which correspond to the nucleic acid
molecules show some structural features of cytokines. In addition
to the nucleic acid molecules and the proteins, various uses of the
molecules are disclosed. The molecules are referred to as T cell
inducible factors.
Inventors: |
Dumoutier, Laure; (Brussels,
BE) ; Louahed, Jamila; (Brussels, BE) ;
Renauld, Jean-Christophe; (Brussels, BE) |
Correspondence
Address: |
FULBRIGHT & JAWORSKI, LLP
666 FIFTH AVE
NEW YORK
NY
10103-3198
US
|
Family ID: |
27391052 |
Appl. No.: |
10/627273 |
Filed: |
July 25, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10627273 |
Jul 25, 2003 |
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09751797 |
Dec 29, 2000 |
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09751797 |
Dec 29, 2000 |
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09354243 |
Jul 16, 1999 |
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6359117 |
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09354243 |
Jul 16, 1999 |
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09178973 |
Oct 26, 1998 |
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6274710 |
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Current U.S.
Class: |
435/6.12 ;
435/320.1; 435/325; 435/6.13; 435/69.1; 530/350; 536/23.5 |
Current CPC
Class: |
C07K 14/52 20130101 |
Class at
Publication: |
435/006 ;
435/069.1; 435/320.1; 435/325; 530/350; 536/023.5 |
International
Class: |
C12Q 001/68; C07H
021/04; C07K 014/705 |
Claims
1. An isolated nucleic acid molecule which encodes a T cell derived
inducible factor, the complementary sequence of which hybridizes,
under stringent conditions, to at least one of SEQ ID NO: 7, SEQ ID
NO: 8, SEQ ID NO: 9, SEQ ID NO: 29, SEQ ID NO: 24 or SEQ ID NO:
25.
2. The isolated nucleic acid molecule of claim 1, wherein said
isolated nucleic acid molecule encodes a protein having the amino
acid sequence of the protein encoded by SEQ ID NO: 7, SEQ ID NO: 8,
SEQ ID NO: 9, SEQ ID NO: 29, SEQ ID NO: 24 or SEQ ID NO: 25.
3. The isolated nucleic acid molecule of claim 1, wherein said
molecule is cDNA.
4. The isolated nucleic acid molecule of claim 1, wherein said
molecule is genomic DNA.
5. The isolated nucleic acid molecule of claim 2, the nucleotide
sequence of which consists of the nucleotide sequence SEQ ID NO: 7,
SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 29, SEQ ID NO: 24 or SEQ ID
NO: 25.
6. The isolated nucleic acid molecule of claim 4, having the
nucleotide sequence of SEQ ID NO: 25.
7. An isolated nucleic acid molecule which encodes the protein
encoded by the isolated nucleic acid molecule of claim 1.
8. Expression vector comprising the isolated nucleic acid molecule
of claim 1, operably linked to a promoter.
9. Expression vector comprising the isolated nucleic acid molecule
of claim 2, operably linked to a promoter.
10. Expression vector comprising the isolated nucleic acid molecule
of claim 3, operably linked to a promoter.
11. Expression vector comprising the isolated nucleic acid molecule
of claim 4, operably linked to a promoter.
12. Expression vector comprising the isolated nucleic acid molecule
of claim 5, operably linked to a promoter.
13. Expression vector comprising the isolated nucleic acid molecule
of claim 6, operably linked to a promoter.
14. Recombinant cell comprising the isolated nucleic acid molecule
of claim 1.
15. Recombinant cell comprising the isolated nucleic acid molecule
of claim 2.
16. Recombinant cell comprising the expression vector of claim
8.
17. Recombinant cell comprising the expression vector of claim
9.
18. Recombinant cell comprising the expression vector of claim
10.
19. Recombinant cell comprising the expression vector of claim
11.
20. Isolated protein encoded by the isolated nucleic acid molecule
of claim 1, and having a molecular weight of about 17-30
kilodaltons as determined by SDS-PAGE.
21. The isolated protein of claim 20, comprising at least 120 amino
acids of the protein encoded by SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID
NO: 9, SEQ ID NO: 29, SEQ ID NO: 24, or SEQ ID NO: 25.
22. The isolated protein of claim 21, comprising at least all but
the 40 N terminal amino acids encoded by SEQ ID NO: 7, SEQ ID NO:
8, SEQ ID NO: 9, SEQ ID NO: 29, SEQ ID NO: 24, or SEQ ID NO:
25.
23. The isolated protein of claim 22, comprising at least all but
the 20 N terminal amino acids encoded by SEQ ID NO: 7, SEQ ID NO:
8, SEQ ID NO: 9, SEQ ID NO: 29, SEQ ID NO: 24, or SEQ ID NO:
25.
24. Antibody which specifically binds to the isolated protein of
claim 20.
25. The antibody of claim 24, wherein said antibody is a monoclonal
antibody.
26. A method for determining effectiveness of interleukin-9 on a
cell, comprising contacting said cell with an agent specific for at
least one of (i) an isolated nucleic acid molecule which encodes a
protein whose amino acid sequence is identical to the amino acid
sequence encoded by the nucleotide sequence of SEQ ID NO: 7, SEQ ID
NO: 8, SEQ ID NO: 9, SEQ ID NO: 29, SEQ ID NO: 24 or SEQ ID NO: 25
and (ii) a protein whose amino acid sequence is identical to the
amino acid sequence encoded by the nucleotide sequence of SEQ ID
NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 29,SEQ ID NO: 24 or
SEQ ID NO: 25 and determining interaction of said agent with (i) or
(ii) as a determination of effectiveness of interleukin-9 on said
cell.
27. The method of claim 26, wherein said agent is an antibody which
specifically binds to (ii).
28. The method of claim 26, wherein said agent comprises the
isolated nucleic acid molecule of SEQ ID NO: 7, SEQ ID NO: 8, SEQ
ID NO: 9, SEQ ID NO: 29, SEQ ID NO: 24, or SEQ ID NO: 25.
29. A method for stimulating activation of a STAT protein
comprising administering an amount of the protein of claim 20
sufficient to stimulate activation of said STAT protein.
30. The method of claim 29, wherein said STAT protein is STAT1 ,
STAT3 or STAT 5.
31. A method of inhibiting activation of a STAT protein, comprising
administering an amount of an antagonist of the protein of claim 20
sufficient to inhibit stimulation of expression of said STAT
protein by said protein.
32. The method of claim 31, wherein said STAT protein is STAT1 ,
STAT3 or STAT 5.
33. A method for determining presence of TIF in a sample,
comprising contacting said sample with an agent which finds to TIF
or a nucleic acid molecule encoding TIF, and determining said
binding as a determination of TIF in said sample.
34. The method of claim 33, wherein said agent is an antibody.
35. The method of claim 33, wherein said agent is a nucleic acid
molecule.
36. A method for screening to determine if a substance influences
IL-9 activity, comprising adding said substance to a sample of TIF
producing cells, in the presence of IL-9, and determining
production of TIF, wherein a difference in production of TIF by
said cells as compared to production of TIF by said cells in
presence of IL-9 but not said substance indicates said substance
influences IL-9 activity.
37. The method of claim 36, wherein said substance is an IL-9
inhibitor or antagonist, said method further comprising determining
lower levels of TIF production by said cells in the presence of
said substance as compared to its absence.
38. The method of claim 36, wherein said substance is an IL-9
activator, said method further comprising determining higher levels
of TIF production by said cells in the presence of said substance
as compared to its absence.
39. A method for determining an aberrant level of IL-9 activity in
a subject, comprising determining level of TIF in a subject and
comparing said level to a normal level, differences therebetween
being indicative of an aberrant level of IL-9 in said subject.
40. The method of claim 39, wherein said aberrant level is excess
endogenous IL-9.
41. The method of claim 39, wherein said aberrant level is
insufficient endogenous IL-9.
42. The method of claim 40, wherein said subject suffers from
asthma, an allergy, or lymphoma.
43. A method for inhibiting IL-9 induced activity in a subject in
need thereof, comprising administering an amount of a TIF inhibitor
sufficient to inhibit IL-9 induced activity.
44. The method of claim 43, wherein said TIF inhibitor is an
antisense molecule.
45. The method of claim 43, wherein said inhibitor is an
antibody.
46. A method for treating a subject suffering from asthma or an
allergy, comprising administering to said subject an amount of a
TIF mutein sufficient to alleviate said asthma or allergy.
47. A method for determining if a mutein of TIF is therapeutically
useful, comprising contacting a cell which produces IL-9 with said
mutein, and determining effect of said mutein on prouction of IL-9,
reduction thereof being indicative of possible thereapeutic
efficacy for said mutein.
48. A method for determining susceptibility to a condition
characterized by aberrant expression of TIF, comprising determining
nucleotide sequence of a TIF gene of a subject believed to possess
an aberrant TIF gene, presence of an aberrant TIF gene being
indicative of possible susceptibility to asthma or allergy.
49. The method of claim 48, comprising contacting a sample taken
from said subject with a pair of oligonucleotide primers which
amplify said TIF gene.
Description
RELATED APPLICATIONS
[0001] This application is a continuation in part of Ser. No.
09/354,243, filed on Jul. 16, 1999, which in turn is a continuation
in part of Ser. No. 09/178,973, filed Oct. 26, 1998. Both of these
applications are incorporated by reference in its entirety.
FIELD OF THE INVENTION
[0002] This invention relates to newly isolated nucleic acid
molecules and their uses. The nucleic acid molecules are shown to
be upregulated by the cytokine interleukin-9 ("IL-9"). Also
disclosed are the proteins encoded thereby. They are described as T
Cell Derived Inducible Factors ("TIFs"). These nucleic acid
molecules encode proteins which induce STAT activation in cells.
They can be used, for example, in the stimulation of regeneration
of targeted tissues. Further, their inhibitors or antagonists can
be used to retard, prevent or inhibit differentiation of other
tissues.
BACKGROUND AND PRIOR ART
[0003] The last decade has seen knowledge of the immune system and
its regulation expand tremendously. One area of particular interest
has been that of research on the proteins and glycoproteins which
regulate the immune system. One of the best known families of these
molecules are the cytokines. These are molecules which are involved
in the "communication" of cells with each other. The individual
members of the cytokine family have been found to be involved in a
wide variety of pathological conditions, such as cancer and
allergies. Whereas sometimes the cytokines are involved in the
pathology of the condition, they are also known as being
therapeutically useful.
[0004] Interleukins are one type of cytokine. The literature on
interleukins is vast. An exemplary, but by no means exhaustive
listing of the patents in this area includes U.S. Pat. No.
4,778,879 to Mertelsmann et al.; U.S. Pat. No. 4,490,289 to Stern;
U.S. Pat. No. 4,518,584 to Mark et al.; and U.S. Pat. No. 4,851,512
to Miyaji et al., all of which involve interleukin-2 or "IL-2."
Additional patents have issued which relate to interleukin-1
("IL-1"), such as U.S. Pat. No. 4,808,611 to Cosman. The disclosure
of all of these patents are incorporated by reference herein. More
recent patents on different interleukins include U.S. Pat. Nos.
5,694,234 (IL-13); 5,650,492 (IL-12); 5,700,664, 5,371,193 and
5,215,895 (IL-11); 5,728,377, 5,710,251, 5,328,989 (IL-10);
5,580,753, 5,587,302, 5,157,112, 5,208,218 (IL-9); 5,194,375,
4,965,195 (IL-7); 5,723,120, 5,178,856 (IL-6), and 5,017,691
(IL-4). Even a cursory review of this patent literature shows the
diversity of the properties of the members of the interleukin
family. One can assume that the larger cytokine family shows even
more diversity. See, e.g., Aggarwal et al., ed., Human Cytokines:
Handbook For Basic And Clinical Research (Blackwell Scientific
Publications, 1992), Paul, ed., Fundamental Immunology (Raven
Press, 1993), pg 763-836, "T-Cell Derived Cytokines And Their
Receptors", and "Proinflammatory Cytokines and Immunity." All cited
references are incorporated by reference.
[0005] The relationships between various cytokines are complex. As
will be seen from the references cited herein, as the level of a
particular cytokine increases or decreases, this can affect the
levels of other molecules produced by a subject, either directly or
indirectly. Among the affected molecules are other cytokines.
[0006] The lymphokine IL-9, previously referred to as "P40," is a
T-cell derived molecule which was originally identified as a factor
which sustained permanent antigen independent growth of T4 cell
lines. See, e.g., Uyttenhove et al., Proc. Natl. Acad. Sci. 85:
6934 (1988), and Van Snick et al., J. Exp. Med. 169: 363 (1989),
the disclosures of which are incorporated by reference, as is that
of Simpson et al., Eur. J. Biochem. 183: 715 (1989).
[0007] The activity of IL-9 was at first observed on restricted T4
cell lines, failing to show activity on CTLs or freshly isolated T
cells. See, e.g., Uyttenhove et al., supra, and Schmitt et al.,
Eur. J. Immunol. 19: 2167 (1989). This range of activity was
expanded when experiments showed that IL-9 and the molecule
referred to as T cell growth Factor III ("TCGF III") are identical
to MEA (Mast Cell Growth Enhancing Activity), a factor which
potentiates the proliferative response of bone marrow derived mast
cells to IL-3, as is described by Hultner et al., Eur. J. Immunol.
and in U.S. patent application Ser. No. 498,182 filed Mar. 23,
1990, the disclosures of both being incorporated by reference
herein. It was also found that the human form of IL-9 stimulates
proliferation of megakaryoblastic leukemia. See Yang et al., Blood
74: 1880 (1989). Recent work on IL-9 has shown that it also
supports erythroid colony formation (Donahue et al., Blood 75(12):
2271-2275 (6-15-90)); promotes the proliferation of myeloid
erythroid burst formation (Williams et al., Blood 76: 306-311
(9-1-90); and supports clonal maturation of BFU-E's of adult and
fetal origin.(Holbrook et al., Blood 77(10): 2129-2134 (5-15-91)).
Expression of IL-9 has also been implicated in Hodgkins's disease
and large cell anaplastic lymphoma (Merz et al., Blood 78(8):
1311-1317 (9-1-90). Genetic analyses of mice that were susceptible
or resistant to the development of bronchial hyperresponsiveness
have unraveled a linkage with the IL-9 gene as well as a
correlation between IL-9 production and susceptibility in this
model (Nicolaides et al., Proc. Natl. Acad. Sci. USA, 94,
13175-13180, 1997). Human genetic studies also point to the IL-9
and IL-9R genes as candidates for asthma (Doull et al., Am. J.
Respir. Crit. Care Med., 153, 1280-1284, 1996; Holroyd et al.,
Genomics 52 233-235, 1998). Secondly, IL-9 transgenic mice allowed
for the demonstration that increased IL-9 expression result in lung
mastocytosis, hypereosinophilia, bronchial hyperresponsiveness and
high levels of IgE (Temann et al., J. Exp. Med. 188, 1307-1320,
1998; Godfraind et al., J. Immunol. 160, 3989-3996, 1998; McLane et
al., Am. J. Resp. Cell. Mol. 19:713-720 (1999). Taken together,
these observations strongly suggest that IL-9 plays a major role in
this disease Additional work has implicated IL-9 and muteins of
this cytokine in asthma and allergies. See, e.g. PCT Application
US96/12757 (Levitt, et al), and PCT US97/21992 (Levitt, et al),
both of which are incorporated by reference..
[0008] IL-9 is known to affect the levels of other molecules in
subjects. See Louahed et al. J. Immunol. 154: 5061-5070 (1995;
Demoulin et al., Mol. Cell. Biol. 16: 4710-4716 (1996), both
incorporated by reference. It will be recognized that the molecules
affected have their own functions in biological systems. For
example, Demoulin et al. show that many of the known activities of
IL-9 are mediated by activation of STAT transcription factors. As
such, there is continued interest in trying to identify molecules
whose presence and/or level is affected by other molecules, such as
cytokines.
[0009] The disclosure which follows describes such molecules. It
was found that nucleic acid molecules encoding the proteins of the
invention were expressed in the presence of IL-9, but not in its
absence. Hence, these molecules are, inter alia, "markers" for the
expression or effect of IL-9 in a subject. The molecules are
referred to as T Cell Derived Inducible Factors or "TIFS"
hereafter. These and other features of the invention will be seen
in the disclosure which follows.
BRIEF DESCRIPTION OF THE FIGURE
[0010] FIG. 1 compares deduced amino acid sequences of murine and
human TIF (SEQ ID NOS: 27 and 28, respectively).
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
Example 1
[0011] The murine lymphoma cell line BW5147 is well known as a cell
line which can be grown in vitro, without the need to add any
cytokines to its culture medium. In order to identify genes induced
by IL-9, samples of BW5147 were cultured either with (200 U/ml), or
without IL-9, for 24 hours. Then, total RNA was isolated, using
guanidium isothiocyanate lysis, and CsCl gradient centrifugation.
These techniques are well known in the art. Following this,
polyadenylated RNA was purified from the total RNA, by using an
oligo(dT) cellulose column. The isolated, polyA RNA was then used
to generate double stranded cDNA. A commercially available
oligo(dT) primer was used. Anywhere from 3-5 ug of polyA RNA were
heated to 70.degree. C. for 10 minutes with 1 .mu.g of oligo dT,
and then incubated with 5.times. first strand buffer (250 mM HCl
(pH 8.3), 375 mM KCl, 15 mM MgCl.sub.2), 10 mM dithiothreitol, 500
uM of deoxynucleotide triphosphates, and 800 U of reverse
transcriptase. Total volume of the reaction mixture was 20 ul, and
the reaction was allowed to proceed at 37.degree. C. for one hour.
This resulted in synthesis of the first stand of cDNA. Second
strand synthesis was accomplished by adding 30 ul of 5 second
strand buffer (100 mM Tris-HCl (pH 6.9)), 450 mM KCl, 23 mM
MgCl.sub.2, 10.75 mM .beta.-NAD+, 50 mM (NH.sub.4).sub.2SO.sub.4,
together with 60 U of E. coli derived DNA polymerase I, 2 U of E.
coli RNase H, 10 U of E. coli DNA ligase, and 250 uM of
deoxynucleotide triphosphates, and brought to a final volume of 150
ul. The mixture was incubated for two hours, at 16.degree. C.
[0012] The product was extracted using phenol-chloroform, and was
precipitated with ethanol. The final cDNA product was then
resuspended in 200 .mu.l of TE.
[0013] These steps were carried out for both the stimulated BW5147
cells ("tester" hereafter), and for parallel, unstimulated BW5147
cells ("driver" hereafter).
Example 2
[0014] The cDNA prepared in Example 1 was then subjected to
subtraction cloning in accordance with well known methods. To do
this, six oligonucleotides were prepared:
1 5'-AGCACTCTCC AGCCTCTCAC CGCA-3; (SEQ ID NO: 1) 5'-GATCTGCGGT
GA-3'; (SEQ ID NO: 2) 5'-ACCGACGTCG ACTATCCATG AACA-3'; (SEQ ID NO:
3) 5'-GATCTGTTCA TG-3'; (SEQ ID NO: 4) 5'-AGGCAACTGT GCTATCCGAG
GGAA-3'; (SEQ ID NO: 5) and 5'-GATCTTCCCT CG-3'. (SEQ ID NO: 6)
[0015] These were used as explained herein. Double stranded cDNA (2
ug), was digested with restriction endonuclease DpnII, extracted
with phenol-chloroform, precipitated with ethanol, and resuspended
in 20 ul of TE (10 mM Tris-HCl (pH 7.5); 1 mM EDTA). Twelve ul (1.2
ug), of cut cDNA was ligated to double stranded SEQ ID NOS: 1 and
2, in a mixture which included 4 ul of desalted SEQ ID NO: 1 (2
mg/ml), 4 ul desalted SEQ ID NO: 2 (1 mg/ml), 10 .mu.l of 5.times.
adapter buffer (330 mM Tris-HCl, pH 7.6, 50 mM MgCl.sub.2, 5 mM
ATP), 7 .mu.l DTT (100 mM), and 28 .mu.l of H.sub.2O). The
oligonucleotides were annealed to each other and to the sample DNA
by heating the mixture to 50.degree. C. and then cooling it to
10.degree. C. over one hour, followed by adding 5 ul of T4 DNA
ligase, and incubation for 12-14 hours, at 12-16.degree. C. The
mixtures were diluted by adding 140 ul of TE. PCR was then carried
out on 200 ul samples, as described infra.
Example 3
[0016] To carry out PCR, 200 ul samples containing 2 ul of the
ligation product in a buffer of 66 mM Tris-HCl, pH 8.8, 4 mM
MgCl.sub.2, 16 mM (NH.sub.4).sub.2SO.sub.4, 33 ug/ml BSA, 0.3 mM of
each dNTP (concentration: 500 .mu.M), and 2 ug of SEQ ID NO: 2 were
first heated at 72.degree. C. for three minutes to remove any of
SEQ ID NO: 1 which was hybridized to the product of Example 2. The
3' ends were then filled in by using 5 U of Taq polymerase (5
minutes, 72.degree. C.). Twenty cycles of amplification were
carried out (1 cycle:1 minute at 95.degree. C., and three minutes
at 72.degree. C.), after which products were combined, phenol
extracted, ethanol precipitated, and resuspended in TE buffer, at a
concentration of 0.5 ug/ul. Hereinafter, this is referred to as the
representation.
Example 4
[0017] The representation was then prepared for subtractive
hybridization by removing SEQ ID NO: 1 therefrom by digestion with
Dpn II. The resulting digest was phenol extracted and ethanol
precipitated. In the case of the unstimulated sample, this resulted
in the driver, while the stimulated sample resulted in the tester.
Portions of tester (20 ug) were gel purified on a 1.2% agarose gel
and isolated. Samples (2 ug), were ligated to SEQ ID NOS: 3 and 4,
in the same way that SEQ ID NOS: 1 and 2 were ligated, as
described, supra.
[0018] In a first cycle of subtractive hybridization, 0.4 ug
samples of tester with SEQ ID NOS: 3 and 4 ligated thereto were
mixed with 40 ug of driver CDNA. The mixture was phenol extracted,
ethanol precipitated, dissolved in 2 ul of 3XEE buffer (30 mM EPPS
pH 8.0), 3 mM EDTA; pH 8.0, 3 mM EDTA. This was overlaid with 30 ul
of mineral oil, and denatured for five minutes at 98.degree. C. A
5M NaCl solution (0.5 ul) was added, and DNA was hybridized for 20
hours, at 67.degree. C. The reaction mixture was diluted to 200 ul
with TE, and tRNA carrier. The samples were incubated for three
minutes at 72.degree. C. to melt away SEQ ID NO: 4, and then four
PCR reactions (200 ul) were prepared. These included 20 ul of
diluted hybridization mix without primer, to fill in the ends of
the reannealed tester, followed by 10 cycles of amplification after
adding samples of SEQ ID NO: 3 (1 cycle: 1 minute at 95.degree. C.,
three minutes at 70.degree. C.) after which products were combined,
phenol extracted, ethanol precipitated, and resuspended in 40 .mu.l
of 0.2XTE buffer. Single stranded DNA was degraded by a 30 minute
treatment of 20 .mu.l of this material with 20 U of mung bean
nuclease, at a total volume of 40 ul. Samples was diluted (1:5), in
50 mM Tris-HCl, at pH 8.9, followed by five minutes of heating at
98.degree. C. to inactivate the enzyme. A second PCR was carried
out, using 20 ul of the product described supra, 2 ul of SEQ ID NO:
3 (1 mg/ml), and 1 ul (5 U) of Taq DNA polymerase. A total of 18
cycles (1 cycle:1 minute at 95.degree. C., three minutes at
70.degree. C.) were carried out. Products were combined, phenol
extracted, ethanol precipitated, and resuspended at 0.5-1 ug/.mu.l.
The product is referred to hereafter as "DP1", or the first
difference product.
Example 5
[0019] DP1 was then digested with endonuclease DpnII, as described
above, and was ligated to SEQ ID NOS: 5 and 6, following the same
processes described for SEQ ID NOS: 1, 2, 3 and 4. Subtractive
hybridization and selective amplification, as described in example
4, was repeated, and second difference product, or "DP2", was
generated. In these experiments, 50 ng of DP1 was the tester. The
driver (40 ug), was as described supra. The process was repeated to
generate a third difference product, using SEQ ID NOS: 3 and 4 as
adapters. To generate the third product, 100 pg of tester were
mixed with 40 .mu.g of driver. All steps of the protocols supra
were repeated, except the final amplification was carried out for
22 cycles, where one cycle was one minute at 95.degree. C., and
three minutes at 70.degree. C. This yielded the final difference
product.
Example 6
[0020] The final difference products were digested with DpnII, and
then cloned into the BamHI site of a commercially available vector,
i.e., ptZ19R. Double stranded DNA plasmids were prepared, and then
sequenced, using standard methods. The sequences were compared to
known sequences in the GenBank and EMBL data bases, using a BLAST
search program.
[0021] At the end of this subtraction procedure, a short cDNA
fragment was identified, i.e., a fragment about 200 base pairs
long. This fragment was used to screen a CDNA library from BW 5147
cells. The largest clone was sequenced. It is discussed infra. It
does not correspond to any known sequence.
[0022] The nucelotide sequence (SEQ ID NO: 7), is 1121 bases long,
including a 537 base pair open reading frame, which encodes a
protein 179 amino acids long. The predicted molecular weight of the
protein is 20,093. There are two additional ATG codons which, if
they acted as start codons, would produce proteins 172 and 167
amino acids in length, with molecular weights of 19,335 and 18,770
daltons, respectively. Each form of the protein is characterized by
a sequence of hydrophobic amino acids which would be cleaved off of
the molecule via the endoplasmic reticulum to provide a mature
protein.
[0023] Analysis of the sequence shows three AT rich motifs
(TTATTTAT). These motifs are often found in 5'-untranslated regions
of cytokines and oncogenes. Kruvs, et al., Science 245: 852 (1989),
have shown that these repeats modulate stability of mRNA for
TIF.
Example 7
[0024] The cDNA isolated and analyzed in example 6, supra, was then
used as a probe to identify genomic DNA for TIF.alpha..
[0025] A genomic library prepared from mouse strain 129 was
screened with SEQ ID NO: 7, following standard methods. An EcoRI
fragment from a positive clone was subcloned into plasmid pZERO and
partially sequenced. The partial sequence is presented as SEQ ID
NO: 8.
Example 8
[0026] A second EcoRI fragment from the positive clone described in
Example 7, supra, was also subcloned. There was a great deal of
homology, but the sequences were not identical. To be specific,
intron 1 of this sequence was 98% identical to SEQ ID NO: 8, intron
2 was 100% identical and intron 3 was 92% identical.
[0027] What is striking about the sequences is that the promoters
are not at all homologous, suggesting independent regulation. The
5' untranslated regions are 92% identical. The first exon for
TIF.alpha. is split into exon la and exon 1.beta.. The first coding
exon (which is exon 1b for TIF.alpha. and exon 1 for TIF.beta.) are
99.5% identical, while the second exons are 100% identical, the
third exons 97% identical, the fourth exons 98.5% identical, and
96% for the fifth exon. In the untranslated 3- region, homology is
96%.
Example 9
[0028] Using the information described in example 8, supra, a cDNA
sequence for the second clone, designated TIF.beta. was deduced,
and is set forth as SEQ ID NO: 9. The genomic DNA sequence was also
ascertained, in the same manner as is described, supra, and is set
forth as SEQ ID NO: 29.
[0029] As compared to the coding region for TIF.alpha., that of
TIF.beta. has six silent changes. There are two changes which
result in an inconsequential amino acid change (at both of
positions 36 and 113, Val in TIF.alpha. becomes Ile in TIF.beta.).
There is also a more significant change, at position 112, where Gln
becomes Arg.
Example 10
[0030] Experiments were undertaken to study expression of the TIFs.
BW 5147 cells were stimulated with recombinant murine IL-9 (200
U/ml), for varying periods of time (0.2, 0.5, 1, 2 & 24 hours).
Total RNA was then isolated, using standard methods and reagents.
Reverse transcription was then carried out, using 5 .mu.g total RNA
and an oligo (dT) primer. Samples of CDNA corresponding to 20 ng of
total RNA were then amplified for 25 cycles using different
primers. (One cycle was 4 minutes at 94.degree. C., 1 minute at
57.degree. C., and 2 minutes at 72.degree. C.). The TIF primers
were:
5'-CTGCCTGCTT CTCATTGCCC T-3' (SEQ ID NO: 10)
[0031] and
5-CAAGTCTACC TCTGGTCTCA T-3' (SEQ ID NO: 11)
[0032] (sense and antisense, respectively).
[0033] These correspond to nucleotides 106-126, and 764-784 of SEQ
ID NO: 7, respectively. As a control, .beta.-actin was amplified as
well, for 18 cycles (first cycle: 4 minutes at 94.degree. C., 1
minute at 60.degree. C., 2 minutes at 72.degree. C. Succeeding
cycles were 1 minute at 94.degree. C., 1 minute at 60.degree. C., 2
minutes at 72.degree. C.).
[0034] Following amplification, post PCR products were analyzed on
a 1% agarose gel, and specific amplification was confirmed,
following blotting, using internal radioactive probes. The probe
for TIF was:
5'-GACGCAAGCA TTTCTCAGAG-3' (SEQ ID NO: 12)
[0035] the conditions and probes set forth were not specific for
one or the other of the forms of TIF; however, the amplification
product of TIF.alpha. contains a KpnI restriction site, while the
restriction site for TIF.beta. does not. Digestion of the
amplification products with KpnI indicated that most, if not all,
of the TIF mRNA induced by IL-9 was TIF.alpha., suggesting that the
TIF.alpha. expression was induced rapidly via the IL-9. The mRNA
for TIF.alpha. was detectable after 30 minutes of stimulation, and
reached a plateau over a 1-24 hour time period.
Example 11
[0036] Experiments were then carried out which showed that the
induction of TIF mRNA by IL-9, described supra, does not require
protein synthesis. In these experiments, total RNA was extracted
from cells stimulated for 24 hours, as described in example 10, but
with or without 10 .mu.g/ml of a protein synthesis inhibitor,
cycloheximide, for 4.5 hours. In a parallel set of experiments,
cells were not stimulated. The total RNA was extracted, and RT-PCR
amplification was carried out as described in example 10. Post-PCR
products were analyzed on an ethidium bromide-stained, 1% agarose
gel. What was seen was that the induction by IL-9 still occurred
when protein synthesis was blocked. Hence, the effect of IL-9 is a
direct effect, not requiring the synthesis of a protein
mediator.
Example 12
[0037] In these experiments, the role of STAT proteins in induction
of TIF mRNA was studied on derivatives of the cell line BW5147. The
first line, BWh9R, expresses wild type human IL-9 receptors. The
line BW-Phe116 is a transfectant with a single mutation (at
position 116), which renders the receptor unable to activate STAT
transcription factors. Still another cell line, Bw-mut6, has a
mutation which renders the receptor unable to activate STAT5, while
retaining the ability to activate STAT1 and STAT3. Finally, cell
line BW-mut7 has a single mutation which renders the IL-9 receptor
unable to activate STAT1 and STAT3, but which retains the ability
to activate STAT5.
[0038] Cell stimulation, isolation of total RNA, reverse
transcription and amplification of cDNA were all carried out as
described in example 10 (Cells were stimulated for 24 hours. Both
human and murine recombinant IL-9 were used). The PCR products were
analyzed on an ethidium bromide stained, 1% agarose gel, as
describe supra.
[0039] The analysis revealed that human IL-9 did not induce
expression in BW-Phe116, suggesting that STAT transcription factors
are implicated. It was found that IL-9 induced TIF expression in
the BW-mut6 mutant, but not the mut7 variant, suggesting that STAT1
or STAT3 are involved, but not STAT5.
Example 13
[0040] The expression of TIF mRNA in normal mouse spleen cells was
then studied.
[0041] Spleen cells from 10-12 week old-Balb/c mice were cultured
for 24 hours in control medium or the control medium supplemented
with 20 .mu.g/ml of LPS (which activates B lymphocytes and
macrophages), or ConA (which activates T cells), or ConA plus 1% of
a blocking antiserum against murine IL-9, with .beta. actin being
used as a control. Purification of RNA, RT-PCR analysis were
carried out as described supra.
[0042] The data indicated that TIF is, at best, very weakly
expressed in resting spleen cells, not induced by LPS, but strongly
induced by ConA. Anti IL-9 antiserum did not affect induction by
ConA, suggesting that its effect is not mediated by IL-9, or is
mediated by other cytokines.
[0043] When the ConA activated spleen cells were analyzed using
sequences of RT-PCR products, it was found that these cells were
expressing TIF.alpha. predominantly, or exclusively.
Example 14
[0044] Further experiments showed that TIF mRNA was expressed even
in the absence of IL-9 induction.
[0045] Spleen cells from 5 week old FVB mice were enriched for T
cells, using a nylon wool column. Then, the cells were stimulated
for 24 hours in medium supplemented with ConA (a T cell activator),
or PMA (which activates PKC in most cells), either with or without
IL-9.
[0046] Total RNA was isolated using standard techniques, and then
ten microgram samples were fractionated via electrophoresis on a
1.3% agarose gel containing 2.2M formaldehyde. The fractions were
then transferred to a nitrocellulose membrane, labeled, and assayed
in a hybridization assay following Van Snick, et al, J. Exp. Med.
169: 363 (1989), incorporated by reference.
[0047] The results indicated that the induction of TIF by ConA was
not modified, and that IL-9 did not induce TIF RNA in PMA activated
spleen cells.
Example 15
[0048] The expression of TIF mRNA in various cell lines was tested.
In these experiments, murine cell lines were stimulated for at
least one day, with a particular cytokine. Specifically, 9T7 is a T
cell lymphoma, which responds to IL-2, IL-4 or IL-9. Cell lines TS3
and TS6 are derived from T helper cell clones, and proliferate in
the presence of either IL-2 or IL-9. MC9 and LI38 are mast cell
lines, which proliferate in the presence of either IL-3 or
IL-9.
[0049] Following stimulation, total RNA was prepared using standard
guanidium isothiocyanate lyses, and CsCl gradient
centrifugation.
[0050] The 9T7 line was then analyzed by Northern blotting, as
described in example 14, while the other lines were assayed using
RT-PCR analysis, as described supra.
[0051] It was found that IL-9 upregulated TIF expression in T
helper cells and mast cells, while IL-2 and IL-3 did not. The 9T7
cell line, however, showed roughly the same level of expression,
regardless of the cytokine, indicating that IL-9 is not mandatory
for TIF expression.
Example 16
[0052] The expression of TIF mRNA in B cell lines was then studied.
The cell lines A20, 70Z/3, and BCL-1 are B cell leukemia cell lines
which grow, in vitro, without cytokines. These cells were
stimulated for 24 hours with IL-4 and IL-9 and total RNA was
isolated, using standard methods. Expression was analyzed by RT-PCR
which was carried out for 35 cycles, followed by blotting and
hybridization, as described supra.
[0053] The results indicated that TIF expression is detectable in B
cells, but is weakly upregulated at best in the presence of IL-9
and IL-4.
Example 17
[0054] Experiments were then carried out to study expression of the
inventive molecules in T helper cell lines. TS2 and TS1 are known T
helper cell lines, derived from T helper cell clones, which
proliferate in the presence of either IL-9 or IL-2 (TS2), and
either IL-9 or IL-4 (TS1). Specifically, TS1 or TS2 cells were
grown in the presence of the listed cytokines for at least 10 days,
after which RNA was extracted using known methods. Expression of
the molecules was studied via RT-PCR (35 cycles), using the
protocols described supra. In TS1 cells both IL-4 and IL-9 induce
TIF expression, but IL-2 does not do so in TS2 cells.
Example 18
[0055] Expression of TIF mRNA in various mouse organs were studied.
Total RNA was prepared from liver, kidney, heart, brain, intestine,
spleen, thymus, lung, muscle and bone marrow, using standard
guanidium isothiocyanate methodologies and CsCl gradient
centrifugation. Forty cycles of RT-PCR were carried out, using the
protocols described supra. Strongest expression was found in thymus
tissue, while less intense signals were found in brain tissue, and
weaker expression in the remaining tissues.
Example 19
[0056] The following experiments describe production of TIF.alpha.,
in 293-EBNA cells.
[0057] Complementary DNA for TIF.alpha. was described supra. It was
subcloned into a commercially available expression vector pCEP-4,
in operable linkage with a CMV promoter. The resulting plasmids
were transfected into 293-EBNA cells, using standard lipofectamine
methods. Following transfection, the cells were incubated in a
methionine free medium, supplemented with .sup.35S labeled
methionine, for 24 hours. Supernatant was harvested, and run on an
acrylamide gel, followed by electrophoresis. The gel was then dried
and exposed to autoradiography for 1 day. A control was then run by
transfecting cells with the same plasmid, in which the cDNA was
cloned in the antisense direction.
[0058] A heterogenous band of about 25-30 kilodaltons was found
from the cells transfected with TIF in the sense direction. Any
discrepancies between the predicted molecular weight, the actual
molecular weight in the system, and the heterogeneity, can be
attributed to glycosylation. In a series of parallel experiments,
cDNA encoding human TIF was expressed in the same way as the murine
cDNA was expressed. With the exception of the change of the CDNA,
all experimental parameters were the same.
Example 20
[0059] Further experiments were carried out to study production of
TIF.alpha. in COS cells. Specifically, TIF.alpha. cDNA was
subcloned into the plasmid pEF-BOS.puro described by Demoulin et
al., supra, in operable linkage with the EF-1.alpha. promoter. The
plasmid cDNA was transfected into COS cells, using the same
lipofectamine method described supra. The cells were incubated in
methionine free medium, supplemented with .sup.35S methionine for
24 hours, after which supernatant was treated as described in
example 20, supra. Again, a heterogenous band of 25-30 kilodaltons
was observed, as well as an 18 kilodalton band, which probably
represents a non-glycosylated form of the molecule.
Example 21
[0060] In these experiments, it was discovered that TIF induces
STAT activation in mesangial, neuronal melanoma, and hepatoma
cells. It is known that when cytokines activate STAT factors, the
factors dimerize, move from cytoplasm to the nucleus, and bind to
target sequences in promoters. The details of the experiments
follow.
[0061] Transfected 293-EBNA cells as described supra were used
following incubation in normal medium for 48 hours, as were
supernatant from the controls, also described supra. Samples of a
mouse kidney mesangial cell line, ("MES13" hereafter), a rat
pheochromocytoma cell line, ("PC12" hereafter), four different
human melanomas (SK23, AUMA, NA-8mel and MULL), human heptaoma
(HepG3) and rat hepatoma (H-4-II-K) were used. Cell samples
(0.5.times.10.sup.6) were stimulated for 5-10 minutes in the
presence of 1% of supernatant. Nuclear extracts were then prepared,
in accordance with Demoulin et al., Mol. Cell. Biol. 16: 4710
(1996), incorporated by reference. In brief, cells were washed with
PBS and then resuspended in 1 ml of ice cold hypotonic buffer for
15 minutes. (Buffer was 10 mM HEPES buffer, pH 7.5, with 10 mM KCl,
1 mM MgCl.sub.2, 5% glycerol, 0.5 mM EDTA, 0.1 mM EGTA, 0.5 mM
dithiothreitol, and 1 mM Pefabloc, 1 mM Na.sub.3V.sub.4, and 5 mM
NaF). Cells were then lysed by adding 65 .mu.l of NP-40, followed
by vortexing. Nuclei were pelleted, by vortexing for 30 seconds at
14,000 rpm, followed by extraction in buffer supplemented with
HEPES (20 mM), glycerol (20%), and NaCl (420 mM). Nuclear debris
was removed by centrifuging for 2 minutes. DNA binding activity was
determined in accordance with Demoulin et al., supra, using a
.sup.32P labeled double stranded oligonucleotide called "GRR,"
which contains the STAT binding site of the Fc.gamma.RI
genepromoter, i.e.:
5'ATGTATTTCC CAGAAA-3' (SEQ ID NO: 13)
[0062] and
5'-CCTTTTCTGG GAAATAC-3' (SEQ ID NO: 14)
[0063] corresponding to the upper and lower strands of the binding
sites in the GRR probe. Briefly, 5 .mu.l volume of nuclear extracts
were incubated in binding buffer (12 mM HEPES, pH 7.6, 10 mM KCl,
0.5 mM EDTA, 2.5% glycerol, 0.1 mg of poly(dI-dC) per ml) for 5
minutes. Radiolabeled GRR probe (10.sup.5 cpm; approximately 0.5
ng) was added, and incubation was continued for 25 minutes before
loading onto a non-denaturing polyacrylamide gel.
[0064] It was also noted that the complexes observed in MES13
cells, described supra, were partially overshifted by both
anti-STAT5 and anti-STAT3 antibodies, showing that (i) the cells
under examination were targets for TIF, and (ii) that STAT3 and
STAT5 are major components of the complex activated by TIF. The
difference in STAT profile, as compared to the profile in Example
12, supra, is attributable to the difference in cell source (human
versus mouse). It was also observed that human TIF works on murine
cells, and vice versa.
Example 22
[0065] This example details the isolation and cloning of a nucleic
acid molecule which encodes human TIF. First, human peripheral
blood mononuclear cells were prepared via standard density gradient
centrifugation. Following this preparation, samples were cultured
for 24 hours, at 3.times.10.sup.6 cells/ml, either with or without
anti-CD3 monoclonal antibody (The antibody was the commercially
available OKT3 mAb, used in the form of ascites fluid at {fraction
(1/500)} dilution). This antibody was used because T cell derived
cytokines are generally expressed only upon activation by e.g., CD3
specific antibodies.
[0066] Total RNA was isolated from these cells, using standard
guanidine-isothiocyanate/CsCl ultra-centrifugation techniques.
Following isolation, 10 .mu.g samples of the RNA were reverse
transcribed using an oligo (dT)15 primer.
[0067] Following preparation of cDNA, as outlined supra, samples
which corresponded to 100 ng of total RNA were amplified, via PCR,
using the following primers:
2 5'-AGCTGCTCAA CTTCACCCTG GA-3' (SEQ ID NO: 15) 5'-CCACTCTCTC
CAAGCTTTTT CA-3' (SEQ ID NO: 16)
[0068] which are based upon a murine cDNA sequence, (i.e., SEQ ID
NO: 7). The PCR conditions involved 30 cycles of amplification,
with one cycle defined as 1 minute at 94.degree. C., followed by 1
minute at 42.degree. C., and then 2 minutes at 72.degree. C.
Amplification product was separated on an agarose gel, using
standard methods, and then sequenced. The result indicated that
fragments of the cDNA had been amplified. Hence, a second reaction
was carried but, using the same materials except SEQ ID NO: 16 was
replaced by SEQ ID NO: 17, i.e.:
5'-CAAGTCTACC TCTGGTCTCA T-3'
[0069] This second PCR reaction was carried out for 25 cycles, with
one cycle being defined as 1 minute at 94.degree. C., followed by 1
minute at 45.degree. C., and then 2 minutes at 72.degree. C. The
amplification product was subjected to the same steps as the first
one. Again, fragments of cDNA were amplified.
Example 23
[0070] Following preparation of amplification product, the 5' end
of cDNA was isolated by using standard, 5'-RACE techniques. In
brief, first strand cDNA was prepared by using SEQ ID NO: 18 as a
primer, i.e.:
5'-TGGCCAGGAA GGGCACCACC T-3'
[0071] This primer was based upon the sequence information obtained
in accordance with example 22. In brief, the 5'-RACE method was
carried out by combining 1 .mu.g of total RNA, prepared as
described supra, 2.5 pmoles of SEQ ID NO: 18, reverse transcriptase
reverse transcriptase buffer, 2.5 .mu.l of dNTP mix (10 mM), 2.5
.mu.l of MgCl.sub.2 (25 mM), and 2.5 .mu.l of dithiothreitol (0.1
M). The reaction was carried out and, after completion, original
RNA was removed via adding RnaseH, and Rnase TI. Any unincorporated
dNTPs, as well as primer and proteins, were removed. The cDNA was
tailed using terminal transferase, or "TdT." This enzyme creates a
3'-binding site for the abridged anchor primer, as described infra.
Tailing was carried out by combining the purified, first strand
cDNA, TdT, buffer (10 mM Tris-HCl, 25 mM KCl, 1.5 mM MgCl.sub.2),
and 200 .mu.M of dCTP.
[0072] Following the tailing reaction, PCR was carried out
using
5'-TGGCCAGGAA GGGCACCACC T-3' (SEQ ID NO: 19),
[0073] and 5'-RACE abridged anchor primer:
5'-GGCCACGCGT CGACTAGTAC GGGIIGGGIIGGGIIG-3' (SEQ ID NO: 20).
[0074] The amplification involved 35 cycles (1 cycle defined as 1
minute at 94.degree. C., 1 minute at 56.degree. C., and 2 minutes
at 72.degree. C.). Following this, nested amplification was
performed on 5 .mu.l of a {fraction (1/100)} dilution of the
amplification product, using SEQ ID NO: 19 and the abridged
universal amplification primer:
5'-GGCCACGCGT CGACTAGTAC-3' (SEQ ID NO: 21).
[0075] Amplification involved 30 cycles (1 cycle being defined as 1
minute at 94.degree. C., 1 minute at 56.degree. C., and 2 minutes
at 72.degree. C.). The resulting PCR product was cloned, following
standard procedures, and sequenced.
[0076] These three protocols, i.e., the two experiments described
supra which generated fragments, and the 5'-RACE PCR, also
described supra, permitted alignment of the sequenced amplification
product, to generate the complete sequence.
[0077] Following the alignment, oligonucleotides were generated
which flanked the deduced open reading frame, i.e.:
5'-CCTTCCCCAG TCACCAGTTG-3' (SEQ ID NO: 22)
[0078] and
5'-TAATTGTTAT TCTTAGCAGG-3' (SEQ ID NO: 23).
[0079] These primers were used to amplify the entire open reading
frame, using mRNA from CD3 specific mAb stimulated cells, as
described supra. For amplification, 25 cycles (1 cycle being
defined as 1 minute at 94.degree. C., 1 minute at 56.degree. C.,
and 2 minutes at 72.degree. C.).
[0080] The complete sequence of the human cDNA is set forth at SEQ
ID NO: 24.
[0081] As with the murine sequence, there are potential start
codons at positions of SEQ ID NO: 24 which correspond to amino
acids 1 and 13, as well as codons corresponding to methionine at
amino acid positions 58, 85, and 92. The possible initiator codons
correspond to proteins with calculated molecular weight of 19,998
daltons, and 18,735 daltons respectively (for 176 or 167 amino
acids, respectively). As with the murine form of the protein,
hydrophobic leader sequences are seen, indicating an N-terminal
signal sequence of from about 20 to about 40 amino acids.
Example 24
[0082] These experiments detail work on the isolation of human
genomic DNA corresponding to the cDNA discussed supra.
[0083] Based upon the cDNA sequences, primers were developed which
correspond to nucleotides 51-70 and the complement of nucleotides
631-650 of SEQ ID NO: 24. PCR was carried out, using standard
methodologies. Specifically, 10 ng of genomic DNA was used as a
template, and 33 cycles of amplification were cararied out (one
cycle of amplification being defined as 94.degree. C. for 30
seconds, 50.degree. C. for 30 seconds, and 72.degree. C. for 5
minutes). Once a sequence was isolated, it was sequenced, and this
is set forth as SEQ ID NO: 25. The sequence is about 4.8 kilobases
in length, and is believed to contain the entire genomic sequence
encoding the TIF molecule, lacking only the 5' flanking region, the
promoter, and the 3' end.
Example 25
[0084] It was of interest to identify where the genomic DNA
discussed supra was located in the human genome. In order to do
this, two different approaches were taken. In the first, the
sequence discussed supra, i.e., SEQ ID NO: 25, was labeled with a
flourescent label, and then was used to probe the human genome via
fluorescent, in situ hybridization ("FISH") using standard
methods.
[0085] In a second approach, a panel of radioactive hybrid clones
were screened using the probe consisting of nucleotides 51-70 of
SEQ ID NO: 24, and 5'-ATCAGATGGA TTACTGAATG-3' (SEQ ID NO:26). PCR
was carried out using 25 ng of genomic DNA as a template, for 35
cycles, where one cycle is defined as 94.degree. C. for in minute,
55.degree. C. for 1 minute and 72.degree. C. for 2 minutes.
[0086] Both methodologies indicated that the gene is located at
chromosome 12q15. Some work links diseases associated with asthma
at this site. See, e.g. Nat. Genet. 15:389-392 (1997); Ober, et al,
Hum. Mol Genet. 7(9):1393-1398(1998); Nickel, et al, Genomic
46(1):159-162(1997); Takahashi, et al, Genomics 44(1):150-2(1997);
Barnes, et al, Genomics 37(1):41-50(1996), all incorporated by
reference..
Example 26
[0087] These experiments describe the manufacture of antibodies
which bind to the TIF protein. To make these, a peptide consisting
of amino acids 40-61 encoded by SEQ ID NO: 7 was coupled to KLH
carrier protein, using standard methods and a ratio of 1 mg peptide
to 1 mg carrier protein. Subject animals (rabbits), were immunized
3 times, at 2 week intervals, with 150 .mu.g of the complex. The
immunogen was emulsified in Complete Freund's Adjuvant for the
first injection, and then Incomplete Freund's Adjuvant for the next
two.
[0088] A first bleed was performed one month after the last
injection, and serum was prepared, following known methods.
[0089] The serum was then tested in a standard Western Blot. In
brief, 10 .mu.l of supernatant from cells transfected with either
SEQ ID NO: 7 or SEQ ID NO:24 were separated via SDS-PAGE
electrophoresis, and then blotted onto PVDF membranes. Antiserum
was diluted to 1:500, and used in a standard Western Blot protocol,
together with anti-rabbit antibody as the secondary antibody, and a
commercially available detection kit.
[0090] It was found that the serum did, in fact, recognize the TIF
protein.
[0091] In FIG. 1, the deduced amino acid sequences of murine and
human TIF are set out. The high degree of homology is seen in the
boxed regions.
[0092] The foregoing examples describe the invention, one aspect of
which are isolated nucleic acid molecules, which encode TIF
proteins such as those with the amino acid sequence of the protein
encoded by the nucleotide sequence of SEQ ID NO: 7, 24 or 25. It
will be appreciated by one of ordinary skill that the degeneracy of
the genetic code facilitates the preparation of nucleic acid
molecules which may not be identical to the nucleotide sequence of
SEQ ID NO: 7, 24 or 25, but which encode the same protein. Of
course, SEQ ID NOS: 7, 24 and 25 are preferred embodiments of this
invention, but other embodiments are also a part of the invention.
Genomic DNA, complementary DNA, and RNA, such as messenger RNA, are
all to be included therein. Isolated nucleic acid molecules from
other animal species, including other mammals, are also a part of
the invention. A preferred aspect of the invention are isolated
nucleic acid molecules whose complements hybridize to SEQ ID NO: 7,
SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 24 under stringent
conditions. "Stringent conditions," as used herein, refer, for
example, to hybridization at 65.degree. C. in buffer
(3.5.times.SSC), 0.02% Ficoll, 0.02% polyvinylpyrrolidone, 0.02%
bovine serum albumin, 25 mM NaH.sub.2PO.sub.4 (pH 7), 0.1% SDS, 2
mM EDTA, followed by a final wash at 2.times.SSC, room temperature
and then 0.1.times.SSC/0.2.times.SDS at temperatures as high as,
e.g., about 65.degree. C. More stringent conditions, such as
0.1.times.SSC, can also be used. These nucleic acid molecules
encode proteins of about 17-22 kD as determined by SDS-PAGE, which
activates STAT proteins, such as STAT1, STAT3 and/or STAT5. In
glycosylated form, these proteins can range from about 17 to about
30 kilodaltons, as determined by SDS-PAGE.
[0093] Also a part of the invention are expression vectors which
include the nucleic acid molecules of the invention, operably
linked to a promoter, so as to facilitate expression of the DNA. It
is well within the skill of the artisan to prepare such
vectors.
[0094] The vectors, as well as the nucleic acid molecules per se,
can be used to prepare recombinant cells, be these eukaryotic or
prokaryotic, wherein either an expression vector or the nucleic
acid molecule itself is incorporated therein. E. coli cells, COS
cells, CHO cells, etc., are all examples of types of cells which
may be used in accordance with this aspect of the invention.
[0095] Proteins encoded by the above referenced nucleic acid
molecules, preferably in isolated form, are another feature of this
invention. By "protein" is meant both the immediate product of
expression of the nucleic acid molecules, glycosylated forms of it,
as well as multimeric forms, such as dimers, trimers, and so forth.
Also a part of the invention are multimers, such as dimers, which
contain at least one protein molecule of the invention, and at
least one, different protein molecule. Preferably, this different
protein molecule is a cytokine, such as IL-10. Also included as a
feature of the inventions are constructs, such as fusion proteins,
where all or a part of the proteins described supra are linked in
some fashion, such as in a fusion protein, to at least one
addtional protein or peptide, or amino acid sequence. The "fusion
partner" may be, for example, a molecule which provides a
recognizable signal, either directly or indirectly, such as a FLAG
peptide, .beta.-galactosidase, luciferase, and so forth. These
fusion partners are preferably joined to the molecule which is
described supra at the N- and/or C- terminus of the protein;
however, it is to be understood that there are many techniques
known for joining molecules to amino acids, and any and all of
these methodologies can produce constructs which are a part of the
invention.
[0096] The individual protein molecules of the invention, as noted
supra, will preferably have a molecular weight of from about 17. to
about 30 kilodaltons, as determined by SDS-PAGE. In multimeric
forms, the molecular weight of the complex will, of course, vary,
but the TIF molecules contained therein will each have a molecular
weight of about 17 to 30 kilodaltons, as determined by
SDS-PAGE.
[0097] The proteins preferably consist of at least about 120 and no
more than about 200 amino acids. Preferably, the amino acids
sequences consists of or comprises all or part of the amino acid
sequences encoded by SEQ ID NOS: 7, 8, 9, 24 or 25. More
preferably, the amino acid sequence contains all but about the
first 40 amino acids encoded by said SEQ ID's. Even more
preferably, it contains all but about the first 20 amino acids
encoded by these sequences. Most preferably, the protein comprises
amino acids set forth at SEQ ID NO: 27 or 28.
[0098] It will be appreciated by the skilled artisan that the
proteins encoded by the above recited nucleic acid molecules are a
feature of the invention, and may be used to produce antibodies, in
accordance with standard protocols. Such antibodies, in monoclonal
and polyclonal form, constitute a further feature of the invention
as do fragments of said antibodies, chimeric forms, humanized
forms, recombinant forms, and so forth. Also a feature of the
invention are immunogens, comprising all or a part of the amino
acid sequence protein molecules of the invention, preferably
combined with an adjuvant, such as Complete or Incomplete Freund's
Adjuvant. Portions of the protein sequences may be linked to other
molecules, such as keyhole limpet hemocyanin, to render them more
immunogenic. These antibodies can be used, e.g., to determine if
the proteins of the invention are present. This is a further
feature of the invention, as is now explained. It has been shown,
in the examples, that the nucleic acid molecules of the invention
were expressed in the presence of the IL-9. Hence, a further
feature of the invention is a method to determine if IL-9 is or has
been present, wherein one detects either the proteins of the
invention, using antibodies for example, or mRNA using the nucleic
acid molecules of the invention, as probes. The mRNA can be
determined directly, or in the form of CDNA. Such probes may or may
not be labeled, as a matter of choice for the user. Hence, one can
determine, for example, if, following administration of IL-9, the
cytokine is still efficacious, by determining if the nucleic acid
molecule of the invention is present. This type of assay can be
adapted, for quantitative studies, wherein one determines, for
example, either if a cell is sensitive to IL-9, and if so, how
sensitive it is. One can also use the proteins of the invention to
phosphorylate STAT proteins such as STAT1, STAT3 and/or STAT 5.
This in turn results in dimerization of the STAT protein, followed
by migration to the nucleus to provoke the effect that these STAT
proteins have on cells.
[0099] One could also use these molecules to test the efficacy of
IL-9 agonists or antagonists when administered to a subject, such
as a subject suffering from lymphoma, an immune system disorder
such as an allergy, acquired immune deficiency syndrome, autoimmune
diabetes, thyroiditis, or any of the other conditions described in,
e.g, U.S. Pat. Nos. 5,830,454; 5,824,551, and pending application
Ser. No. 08/925,348, filed on Sep. 8, 1997 now allowed, all of
which are incorporated by reference. The molecules can also be used
to mediate the role of IL-9 in these and other conditions. To
elaborate, since IL-9 induces TIFs, the TIFs are useful as IL-9
activity mediators. Thus, a further aspect of the invention is a
method to determine activity of endogenous IL-9, such as in
situations where excess IL-9 activity is implicated, such as
asthmas, allergies, and lymphomas. One can also block or inhibit
IL-9 activity by blocking or inhibiting TIF or TIF activity, using,
e.g., antisense molecules, antibodies which bind to TIF, or other
antagonists of in these molecules. For example muteins of TIF,
which bind to the TIF receptor but do not activate it, therby
inhibiting IL-9 induced activity, are a feature of the invention.
Examples of conditions which can be treated by the use of such TIF
muteins are allergies, asthma, and so forth. Muteins in accordance
with the invention can be made in accordance with, e.g., Weigel, et
al, Eur. J. Biochem 180(2):295-300(1989) and Epps, et al, Cytokine
9(3):149-156(1997), both of which are incorporated by reference.
Such muteins can be used in the treatment of asthma, allergies, or
both. Further, it will be clear to the skilled artisan that the
models set forth, supra, can also be used to screen for appropriate
muteins/ The ability to regulate IL-9 activity is important in
conditions such as those listed supra, as well as conditions such
as apoptosis, including cortisol induced apoptosis, conditions
involving the nuclear expression of BCL-3, since IL-9 is known to
induce such expression, and so forth. "Antibodies," as used herein,
refers to any portion of an antibody which binds to TIF, including
chimeric and humanized antibodies.
[0100] Another feature of the invention relates to the ability of
the TIF type molecules of the invention to either promote
regeneration or inhibit differentiation of tissue types on which
the molecules are active. As was shown, supra, the TIF molecules
target various cancer and normal cell lines (i.e., mesangial and
neuronal cells, as well as melanoma and hepatoma cells). Hence, one
can stimulate regeneration of tissue via, e.g., adding an amount of
a TIF type molecule to a sample in need of regeneration of a tissue
acted on by the TIF molecule. This approach can be used both in
vitro, and in vivo. Similarly, antagonists of TIF may be added when
the situation is one where the aim is to inhibit differentiation of
a particular type of tissue, such as melanoma or hepatoma.
[0101] The genes which encode TIF, as noted in Example 25, supra,
are located on chromosome 12. This chromosome is associated with
asthma, as is known in the art. Hence, a further embodiment of the
invention is a method for determining susceptibility to conditions
such as, or related to asthma, by determining if aberrations, such
as polymorphisms, deletions, additions, etc., are present at the
site of the TIF gene. Such aberrations may be an indicia of
susceptibility to, or of the presence of, asthma, an allergic
condition, or one or more related conditions. The ability to detect
aberrations in a DNA sequence is well known in the art, and such
methods need not be set forth herein. Preferably, the aberration or
aberrations is detected via standard techniques, such as PCR, using
the methodologies and primers referred to supra.
[0102] Other features of the invention will be clear to the artisan
and need not be discussed further.
[0103] The terms and expressions which have been employed are used
as terms of description and not of limitation, and there is no
intention in the use of such terms and expressions of excluding any
equivalents of the features shown and described or portions
thereof, it being recognized that various modifications are
possible within the scope of the invention.
Sequence CWU 1
1
29 1 24 DNA Mus musculus 1 agcactctcc agcctctcac cgca 24 2 12 DNA
Mus musculus 2 gatctgcggt ga 12 3 24 DNA Mus musculus 3 accgacgtcg
actatccatg aaca 24 4 12 DNA Mus musculus 4 gatctgttca tg 12 5 24
DNA Mus musculus 5 aggcaactgt gctatccgag ggaa 24 6 12 DNA Mus
musculus 6 gatcttccct cg 12 7 1119 DNA Mus musculus 7 taaacaggct
ctcctctcac ttatcaactg ttgacacttg tgcgatctct gatggctgtc 60
ctgcagaaat ctatgagttt ttcccttatg gggactttgg ccgccagctg cctgcttctc
120 attgccctgt gggcccagga ggcaaatgcg ctgcccgtca acacccggtg
caagcttgag 180 gtgtccaact tccagcagcc gtacatcgtc aaccgcacct
ttatgctggc caaggaggcc 240 agccttgcag ataacaacac agacgtccgg
ctcatcgggg agaaactgtt ccgaggagtc 300 agtgctaaag atcagtgcta
cctgatgaag caggtgctca acttcaccct ggaagacgtt 360 ctgctccccc
agtcagacag gttccagccc tacatgcagg aggtggtacc tttcctgacc 420
aaactcagca atcagctcag ctcctgtcac atcagcggtg acgaccagaa catccagaag
480 aatgtcagaa ggctgaagga gacagtgaaa aagcttggag agagtggaga
gatcaaggcg 540 attggggaac tggacctgct gtttatgtct ctgagaaatg
cttgcgtctg agcgagaaga 600 agctagaaaa cgaagaactg ctccttcctg
ccttctaaaa agaacaataa gatccctgaa 660 tggacttttt tactaaagga
aagtgagaag ctaacgtcca tcatcattag aagatttcac 720 atgaaacctg
gctcagttga aaaagaaaat agtgtcaagt tgtccatgag accagaggta 780
gacttgataa ccacaaagat tcattgacaa tattttattg tcactgatga tacaacagaa
840 aaataatgta ctttaaaaaa ttgtttgaaa ggaggttacc tctcattcct
ttagaaaaaa 900 agcttatgta acttcatttc catatccaat attttatata
tgtaagttta tttattataa 960 gtatacattt tatttatgtc agtttattaa
tatggattta tttatagaaa cattatctgc 1020 tattgatatt tagtataagg
caaataatat ttatgacaat aactatggaa acaagatatc 1080 ttaggcttta
ataaacacat ggatatcata aaaaaaaaa 1119 8 7445 DNA Mus musculus 8
gtctatcacc tgcttaagat tcttctaatt tataaaaaaa actatttctt aaaatgaaaa
60 gcaaccagag cacgtattta tagcatggtg ttctgaccat gcaggtacag
agtggaatgg 120 taagaggcgc tattatcagc attaaccaac atgttaatgt
tttcttctgg caagcaaact 180 tgaaatctat gtcttaaaca atcttcaagc
ctctaatata gtgctaacga ctggagtccg 240 ctgctgtcca acagagctct
tgagcacgct ctcctctgtt tgcaatttta tgttctttga 300 tcgactcccc
aacctctcac cttcggctcc tgatggccac ctttcaactt tctgcattta 360
tgaactccat gttttaatct ttttattaaa atattcacac aatcagtgtt tgtgcaagtc
420 tgtttcaccc acatgtatgt ctgtgcacca agtgctgcct ggtgcttgtg
ggggcaagga 480 gcaggagagg gtgccctggc accggagtca cggatggttg
tgagccacca tgaggatgct 540 gggagttaga cccaggtcct ccagaagtgc
agcaaatgct cttaaccaca cgcaggcatt 600 tctctctcca gccccaacat
gagtgctttt agattccacc tagaatagag atctgatggc 660 ttcactcact
gccacctccc ctttgcatct ttctgccaag gaacaccaaa aagcaagaat 720
ccccacactg ctttcgctcc tcaagtctgc acctctcaac aggtcaagat tctccagtgt
780 ccctctaaca ctttccccag tgtccctcta acactttctc cagtgtccct
ctaacacttt 840 ctccagtgtc cctctaacac ttttgatctc aattagctga
ggggagaaag atctcacaca 900 gtgattttca tgacttcgcg ttctagtcta
gatgtaggca tttgcgtgtc agtctagggt 960 aggcgtctgc tcccgctgct
taggaaagac tttcctagtc tagttgtcag gtgctatctg 1020 ggattcagtg
tacatacaat gcaaaaaatc ccagtatttt gtaaattctc ttcttcaact 1080
atccatctat atagtatgtt attgtaggct catttaaaaa taatattttg agacttatgc
1140 ttgcacaagt aaaatgtcag agaattagca aatgtatagt attattttat
tttaaaaaaa 1200 tctatgctta aaatgtctat tagattgttc actaccgata
tttccaaact taacttgacc 1260 ttggctatga tttcaacctt tgtatttgca
tctaccataa cagtctctga accagaacat 1320 tctgtggcaa tgggagctgt
gaagaaagcc aacattctta ttaaaaaaaa aaaacagcta 1380 gttatagttt
aggattccat atactaaaaa aaatagagat ataattattt taaaaattga 1440
aataatctcc aagttttcat tatggcttat ttcaaagcac agaatatagg acacgggtct
1500 tttatttctg gtcacttcta aagagataag aatctatgaa gttggtggga
aaatgagtcc 1560 gtgaccaaaa cgctgactca atagctacgg gagatcaaag
gctgctctac tcaatcagaa 1620 tctactacgg caaagccatg gctttctttg
aaaaccgtgt ttagaagatt tctgggattt 1680 gtgtgcaaaa gcaccttgtt
ggccctcacc gtgacgtttt agggaagact tcccatctct 1740 caaggtggga
aggcttggag gtggtgtctt gtggcctcct atggtggtta ggtacttctc 1800
agaagacagg actggaaatt agataatgtc tgatgtcata tcattcacaa taccaaaaaa
1860 accctggtgt cccgatggct ataaaagcag caacttctgc ctctcccatc
acaagcagag 1920 acacctaaac aggtaagcac tcagacctct acagacaatc
atctgcttgg taccatgcta 1980 cccgacgaac atgctcccct gatgtttttg
ccttttgctc tctcactaac aggctctcct 2040 ctcacttatc aactgttgac
acttgtgcga tctctgatgg ctgtcctgca gaaatctatg 2100 agtttttccc
ttatggggac tttggccgcc agctgcctgc ttctcattgc cctgtgggcc 2160
caggaggcaa atgcgctgcc cgtcaacacc cggtgcaagc ttgaggtgtc caacttccag
2220 cagccgtaca tcgtcaaccg cacctttatg ctggccaagg aggtacagct
gcatctcttt 2280 ctctccatac cgccttgcca ttttctctga agcacttgca
aactctttag gggcgcttta 2340 tctccgcagg tctcactacc tatgttttct
gtctctttag agactcttta aggactgggt 2400 ctttttctat ttctatttca
aggtctcagg accatttcct atcttggcct tcaggacaca 2460 tatactgaat
tttatctaca gaggcgcatt tagaaagcca cccacgactg caatactttc 2520
catttctctg tgctctcttc tgaactcata ctctcttggc tactcctgag acccactgcg
2580 gacatacatc tctacttaca ggcttttctt ccatctcctt gtcacccagg
cacttagggt 2640 tttctctctt tcaggccagc cttgcagata acaacacaga
cgtccggctc atcggggaga 2700 aactgttccg aggagtcagt gtaagtcctc
actgtgatga gcagggctag ctgcgggagc 2760 tggtggaccc tctgggatag
tctgacgtat gacccctgct gcttcttgtc tacctgcagg 2820 ctaaagatca
gtgctacctg atgaagcagg tgctcaactt caccctggaa gacgttctgc 2880
tcccccagtc agacaggttc cagccctaca tgcaggaggt ggtacctttc ctgaccaaac
2940 tcagcaatca gctcagctcc tgtgtaagtc tgactctggc tacctatgct
cctctctctt 3000 cctcttctat tccagtaaga acccgaggtc ctgccctctc
tctcttcaca agagtgagga 3060 gggcctcagc accaccacca tcataggcca
cttgaaatag gtcacaaagg ctttggcttc 3120 aattgagtaa tactttgagt
ttgtatgagt gaagctttat ttgttttatc catggaaaga 3180 aatcaactca
aattctgtag gatgagaaag atgttgggaa cgaaaaaagg cctagataga 3240
gaaacagatc tgctgagtat agtacttatg gggggagcag ggggcgatat ccactgagta
3300 caagtacttg tggggagaga aatccactga gtacaagtac ttgttggcat
ggagatccac 3360 tgagtacaag tacttgtggg gggagggaat ggcacagagc
aaaagttgaa gggaaggaag 3420 atggagaggc ctcatggttg ggggtgtgaa
aggtcactcc ttttccatgt gatggagagt 3480 taagaaaaac cagtgtgtga
gtttgatgtc ttcagacacc cccaactatg aaacatatcc 3540 acgaggagcg
ggcagactgt gggagacctg gcatttaggg aaggcgcggc ttttcacacg 3600
agaaacttta tgctcatctc ttgtgctaca ctcccacctt tgatgaggtt cagctcaggt
3660 ttcgtttcta ccgttcttgc tactggtgga aacttcagta ggattcccca
aagacgagga 3720 cagctcttct gtaagggagg gacctggatt tcagtgtcct
agagaacgaa atagctcaga 3780 gaatctaggt caacgtgaaa tctaggtcac
agcgggcaaa aatgactgaa cgcctctatt 3840 ccaggtgaac ggtcacgtgc
ctcagatata ctgaggtatt gggctcccac cggataagat 3900 tctgttagtg
agtctgcttt tattttgcag cacatcagcg gtgacgacca gaacatccag 3960
aagaatgtca gaaggctgaa ggagacagtg aaaaaggtac tattggcaag ccacaatact
4020 aagccattca gtaggagacg tggggatttc tttctctgct tcccagtccc
ttctactttg 4080 taacatttta tttgacttgt ctactatctg gtccattact
cgcttagctg cacctgtatc 4140 tagctgggtc tatagatctt tcaatctgtg
tctaaatttg taagtcacaa ttctggagct 4200 agcagaaagc ttagctcagc
cagtctcatg agcacttgct cggaggatgg cttgtgacag 4260 agtcaatgct
agaagacagc atccctgatt cccagctctg cacttgccta gtggccatgt 4320
gtaattactt tggcttgatt aagtatttgg gaaagccagt tcccacggac ctacataatc
4380 tgaagaacca tgcattgaaa actagaaagc tgggcacaaa cttactagag
atgatttttg 4440 agctcattaa acggatgctc tgaaatgtgg caaaatcaac
ccagaataac aacaaaagag 4500 ctggatttgc aaataggaca agtatttaga
atcactggta ttaatagcta tcatcttaat 4560 taaaatatag ggcctatata
tatatttaag attaaacaca agagtggata gcctcccaat 4620 ttacttggcc
tggtttcaaa agagtaaaaa tatcagtcat ggattaatta tagtgtcatg 4680
aaagtatgag atggaaaccc tttccttact ttttaccttc atttcttagt tttttttttc
4740 ttcacaccct gatcaagcca ctagtaagca cctatctgct gtgagctatt
atatgacttt 4800 acagcaaaca acattgctgt gtggcctctt tggggaaggg
aacaggatag caggaggctc 4860 aggctagcaa gtctgacttg ccctaaagcc
agaggcatgg ttgatagcag agaaagtgag 4920 gctcttcgca agtgggtgtg
cttaagtaat cagaaacagg aaggctccgg ttgatggaat 4980 tatcagtaag
atatctaccc ttatctcctt ctatcgaacc taaatcgtct ctttttcttg 5040
tgtgtaggct gataaacaca cttgttttct tttgagtgtt catggctttg tagattttta
5100 gtgctctgcc agttcttgtt agagggtttg ttaccttgac acctgggctt
ggatgttagc 5160 atgccaaagg cacacacttc tgaatgcctg tgtaaaaggt
tattattcat ttactttgtc 5220 tttggaaagg tgaagcgtgt gtgagaaaga
actcacagga gatgtgttct ctgtaggaaa 5280 actttttttt tccccttaaa
tgcctataat ccactttcag tcaactttga cttttatacc 5340 atgctgtcac
atgaaagagt gtttaggccc gctctcatgg ctctgggaaa agcaccaata 5400
ggggaaggaa tgttatgctg agaaatctga ccggcaggga aactggtcag agctcccccg
5460 aagaccacca caggtgttaa gtaggaacag tccagggtgg gctcatgtaa
tagaatggaa 5520 cagagcgagg gaagataagc tacaaagttt catagggtcc
ggagtcttaa agatacaaaa 5580 tagctgcttg ggcttcataa caaaggaagt
ctgggaaggc agcaagtgag agggaaatgg 5640 aaagggaaaa aacagaatgt
agaggacttg aacagctaca aatcctctac cagacgattt 5700 ttcttggaac
aatctagaag gtagtggatt aggtgattgc agggggactt gctttgccat 5760
ttgaatctgg gtttttgtct ctccattgag gttgaaagcg tcaccctttt taccctcgaa
5820 tggaggagga aagaaggggt gttatgactc ctacctggag ttttactagt
ttacgcaatg 5880 gaacagacac tcgggacctc ctcttgacaa aaaaaatgga
aacctgttgt ttgtcttgtt 5940 tgttcttttg ttaagaaagc acaggcaaag
cccgaccaca tgggttgaat gtgggtcttt 6000 gagtcaaggc ttttgagttg
agcactcatc aatagttgat catggtcagg tggagggcta 6060 cctgtcaggc
cgagccctgc tggcttcgca cttaacatct ccaggtctca gtatcacttc 6120
ctgctactta gcacagttag gagttgagca aacctttttt tccaaccccc actaaaattt
6180 aattgacaaa agactgtgta atttgtggga tacagtgtga taattgatct
atgtgtgcat 6240 tgtgcaaggt tcaataagat agattaatag gcccatcaac
agctttatgg gtgtgaaatg 6300 caagtaatat aggtagatgc ctgtggtgtc
cttaggtcag aaaggcatga ttttaaggtc 6360 ttgggcaaat catattatac
tcatgctaaa aatacattat gttgattatt aatcttttag 6420 agaaggctga
tacttggttt tggtgctcag caagcaaatg tcaccagctc tttctaactg 6480
gtaccacttt agaaaatgct acctgtgctc aaattggttt gtattcttat tttcatagct
6540 tggagagagt ggagagatca aggcgattgg ggaactggac ctgctgttta
tgtctctgag 6600 aaatgcttgc gtctgagcga gaagaagcta gaaaacgaag
aactgctcct tcctgccttc 6660 taaaaagaac aataagatcc ctgaatggac
ttttttacta aaggaaagtg agaagctaac 6720 gtccatcatc attagaagat
ttcacatgaa acctggctca gttgaaaaag aaaatagtgt 6780 caagttgtcc
atgagaccag aggtagactt gataaccaca aagattcatt gacaatattt 6840
tattgtcact gatgatacaa cagaaaaata atgtacttta aaaaattgtt tgaaaggagg
6900 ttacctctca ttcctttaga aaaaaagctt atgtaacttc atttccatat
ccaatatttt 6960 atatatgtaa gtttatttat tataagtata cattttattt
atgtcagttt attaatatgg 7020 atttatttat agaaacatta tctgctattg
atatttagta taaggcaaat aatatttatg 7080 acaataacta tggaaacaag
atatcttagg ctttaataaa cacatggata tcataaatct 7140 tctgtcttgt
aatttttctc cctttaatat caacaatacc atcatcatca tcattaccca 7200
atcattctca tgatttcatg cttgacccat attatactgt taaagttggt tcctggaggc
7260 ctgtggtttt gtgtgtgttg tgtgtgtgtg tggggttatg catgtgaaag
ccagagatgg 7320 atattaggtg ttcttctcta tcagtctttg ccttattatt
tgagacaggg tctgtcactg 7380 aacctgtagc taggctggcc aacaagctct
attaattttt tttaagatta attaattatg 7440 tgtat 7445 9 1111 DNA Mus
musculus 9 aacaggctct cctctcagtt atcaactttt gacacttgtg cgatcggtga
tggctgtcct 60 gcagaaatct atgagttttt cccttatggg gactttggcc
gccagctgcc tgcttctcat 120 tgccctgtgg gcccaggagg caaatgcgct
gcccatcaac acccggtgca agcttgaggt 180 gtccaacttc cagcagccgt
acatcgtcaa ccgcaccttt atgctggcca aggaggccag 240 ccttgcagat
aacaacacag acgtccggct catcggggag aaactgttcc gaggagtcag 300
tgctaaggat cagtgctacc tgatgaagca ggtgctcaac ttcaccctgg aagacattct
360 gctcccccag tcagacaggt tccggcccta catgcaggag gtggtgcctt
tcctgaccaa 420 actcagcaat cagctcagct cctgtcacat cagtggtgac
gaccagaaca tccagaagaa 480 tgtcagaagg ctgaaggaga cagtgaaaaa
gcttggagag agcggagaga tcaaagcgat 540 cggggaactg gacctgctgt
ttatgtctct gagaaatgct tgcgtctgag cgagaagaag 600 ctagaaaacg
aagaactgct ccttcctgcc ttctaaaaag aacaataaga tccctgaatg 660
gactttttta ctaaaggaaa gtgagaagct aacgtccacc atcattagaa gatttcacat
720 gaaacctggc tcagttgaaa gagaaaatag tgtcaagttg tccatgagac
cagaggtaga 780 cttgataacc acaaagattc attgacaata ttttattgtc
attgataatg caacagaaaa 840 agtatgtact ttaaaaaatt gtttgaaagg
aggttacctc tcattcctct agaagaaaag 900 cctatgtaac ttcatttcca
taaccaatac tttatatatg taagtttatt tattataagt 960 atacatttta
tttatgtcag tttattaata tggatttatt tatagaaaaa ttatctgatg 1020
ttgatatttg agtataaagc aaataatatt tatgataata actatagaaa caagatatct
1080 taggctttaa taaacacatg aatatcataa a 1111 10 21 DNA Mus musculus
10 ctgcctgctt ctcattgccc t 21 11 21 DNA Mus musculus 11 caagtctacc
tctggtctca t 21 12 20 DNA Mus musculus 12 gacgcaagca tttctcagag 20
13 16 DNA Homo sapiens 13 atgtatttcc cagaaa 16 14 17 DNA Homo
sapiens 14 ccttttctgg gaaatac 17 15 22 DNA Homo sapiens 15
agctgctcaa cttcaccctg ga 22 16 22 DNA Homo sapiens 16 ccactctctc
caagcttttt ca 22 17 21 DNA Homo sapiens 17 caagtctacc tctggtctca t
21 18 21 DNA Homo sapiens 18 tggccaggaa gggcaccacc t 21 19 21 DNA
Homo sapiens 19 tggccaggaa gggcaccacc t 21 20 36 DNA Homo sapiens
24,25,29, 30,34,35 n is inosine 20 ggccacgcgt cgactagtac gggnngggnn
gggnng 36 21 20 DNA Homo sapiens 21 ggccacgcgt cgactagtac 20 22 20
DNA Homo sapiens 22 ccttccccag tcaccagttg 20 23 20 DNA Homo sapiens
23 taattgttat tcttagcagg 20 24 690 DNA Homo sapiens 24 tgcacaagca
gaatcttcag aacaggttct ccttccccag tcaccagttg ctcgagttag 60
aattgtctgc aatggccgcc ctgcagaaat ctgtgagctc tttccttatg gggaccctgg
120 ccaccagctg cctccttctc ttggccctct tggtacaggg aggagcagct
gcgcccatca 180 gctcccactg caggcttgac aagtccaact tccagcagcc
ctatatcacc aaccgcacct 240 tcatgctggc taaggaggct agcttggctg
ataacaacac agacgttcgt ctcattgggg 300 agaaactgtt ccacggagtc
agtatgagtg agcgctgcta tctgatgaag caggtgctga 360 acttcaccct
tgaagaagtg ctgttccctc aatctgatag gttccagcct tatatgcagg 420
aggtggtgcc cttcctggcc aggctcagca acaggctaag cacatgtcat attgaaggtg
480 atgacctgca tatccagagg aatgtgcaaa agctgaagga cacagtgaaa
aagcttggag 540 agagtggaga gatcaaagca attggagaac tggatttgct
gtttatgtct ctgagaaatg 600 cctgcatttg accagagcaa agctgaaaaa
tgaataacta accccctttc cctgctagaa 660 ataacaatta gatgccccaa
agcgattttt 690 25 4797 DNA Homo sapiens 25 tgcacaagca gaatcttcag
aacaggttct ccttccccag tcaccagttg ctcgagttag 60 aattgtctgc
aatggccgcc ctgcagaaat ctgtgagctc tttccttatg gggaccctgg 120
ccaccagctg cctccttctc ttggccctct tggtacaggg aggagcagct gcgcccatca
180 gctcccactg caggcttgac aagtccaact tccagcagcc ctatatcacc
aaccgcacct 240 tcatgctggc taaggaggta tacatctcaa tcctgctctt
tctcgttgga tctacttgga 300 atccaaatag ttcttaaact tttcttcaga
gcatctctaa gagctttagg aacccactgt 360 ttatccctga gggtagataa
attttctgtt ttttcagaga ctctttggga atctggcttt 420 ttttttttct
tgaacttctt ccttccattt tggcctttat gatacatatg atgaattttt 480
cccaaagagc ggccattcag taatccatct gatgattttt ttttccttta tgcctctgtg
540 cattgttcta aactcatgca cacatctgaa ttctgctttt agtctttatg
atgttgctct 600 ggggagacgg gatggggcac atgtctatgt ataaattttt
tttctatttg ctcaatgtcc 660 agacccttag tcttttcttc tcttccaggc
tagcttggct gataacaaca cagacgttcg 720 tctcattggg gagaaactgt
tccacggagt cagtgtaagc tacagttgtg acgaacaggg 780 ccgtgtgccg
tccatgggta cttggggtgg tggtgatgat ggtttaggtc ttatccctta 840
tgaccctttc tgtttccctt ccacctgcag atgagtgagc gctgctatct gatgaagcag
900 gtgctgaact tcacccttga agaagtgctg ttccctcaat ctgataggtt
ccagccttat 960 atgcaggagg tggtgccctt cctggccagg ctcagcaaca
ggctaagcac atgtgtaagt 1020 tcagctctca gcctatgccc acctacccct
ccttccctcc ttccacagag acccccttac 1080 cccaactctc tctccttccc
cctaccccta agctagcagg aagaagtgtc ttggcagcag 1140 tgttatcagg
agtcatttgg gatcatagag tatttgcttt tgctttgact gagtcacatc 1200
ttgagtttat agtggtgaat ggggtctgga acttaagtgt acagaagccg cattggtttg
1260 tcttcggaaa aaaggcaact caggttgcgt aagatgagaa aggtgttggg
aaaacatcta 1320 gctgtggaaa tggatccatt gagtctaagt tgttgagggg
aggggatggc atggagagaa 1380 attagaagag aaagtgggaa atgggaaggc
ttaaagtcgg tggtgggtcg gcagactgtt 1440 gccctgttga tgtcatggga
agccacaaaa tcggaggcgt gtgaacttga tgccgctgaa 1500 catttgaaac
tatgaaaaaa agtttgagtg gagtgggccc agtaaaaggc cctaggactt 1560
actgaagagg gcttaatttt cacatgagat gttttatgta catttcttgt tctaagcatg
1620 caattttctg gagatacgat tgaggtttta ttccttacag aatttgcata
aactactccg 1680 ctctttccac aaatgcaaac ctcagtagga tttcccaaag
atgaagagag gtctcttgta 1740 agggaagtga ctggattctg gcgtccaagg
gaattcaaga gctcaggaaa tctaggtcac 1800 tgttgaaatc taggtcattg
tgggcaaaat tactaagagc tttaattcca ggtgaattgt 1860 actgtacctc
catgggtgtg gaggttcata aagtttcagc acaacattaa gatagttatg 1920
cttgttattg ttttatagca tattgaaggt gatgacctgc atatccagag gaatgtgcaa
1980 aagctgaagg acacagtgaa aaaggtagga ctgataactg tcaatgctaa
gtcatgcaat 2040 aggagagaca aatgttgttt ttctttcctt tctttcttcc
catcactttg tgatttttca 2100 cttgattctc ctaccaccag ggcgattact
ttggtgtctg tgtatgtaga tatatctata 2160 tatctagatg tcagtttcca
aatcttgcaa attgtagaat tctagaactg gttgggatct 2220 tagcttgtct
agtcacataa cctcagattc tggggatggt cagtggcaga gatagggcta 2280
gaatgcaggt ctcctgaatc ccaagccagc acttttcccg gtggtgatac agattagttt
2340 tggtaccatt aattcttagg gaaatttcag attcctattg actcatgtaa
tctgaagaag 2400 tacttgttta aaaacagaaa aatgcctatg ggcaaattta
tttgaagtca tttttgaagt 2460 cattaatgca ttgctttgaa acttggaaga
ataaactcag aacaatgaga aaagagctgg 2520 acttgcatat agggctaatt
tctggagtaa taaacactta ttttgaatta tcataatatc 2580 tatcagatat
tgattatagt ttaaaagcaa gagcagacaa ccccgatctc ttttatacag 2640
gttcaaatag agtaaaaata ttagtaagag atttattata gttaaatgga agtctgaatt
2700 ggtaagcttt tttttcttcc tctctcccat caagaccttc cattctagtt
tcttccttca 2760 ctccctcaac aaatccctag ggagcattta tccatggtgg
gctggtgtac atttctatag 2820 tgaatgatac catcatgtgg cctatttggt
gaaaagaaca acaatggaag gcttagacta 2880 acaatagtga ctcaccccaa
aaccggagga atgattagga gcagtgaaag tgacgctctt 2940 gcaagcaggt
acaactaaat actcagaaac atgaaggctc cagttgatgg aattttcagt 3000
aacaagctta accttaattc cccctttttc cctcttgact ttttaaaaaa gcgtttcttc
3060 ctgagcatca tttaatgagt gtgactgttt cttcctttga taattgaagg
ctttgtagtt 3120 ttaaattgtg aagcccagtt ctcttgttat agaactatta
tctagacatg gagggctgaa 3180 tgttagcatg ccacagacaa ggcatgcttt
acacatcttg cttaaaaaat tactgatttc 3240 atcttgcttg ttgtctttag
aaaagtgaag tgtgagagag gagaatctca tggtgatctg 3300 tgtgattttc
aagaccttta atccattttg aaagaatcaa tttcatattt gcaatgggtt 3360
gccatgtgga agagtgatta tgcttttttg ctggtagctt cagaaagcac aggagggaga
3420 gcaatgttgt tcagagaaag atcaacagga ggagaaactg tcagagctgt
ctgaaatagg 3480 gtggttttgg gaggcattaa ttccctctcg ttgggggtaa
aagcagaacg caggttggta 3540 gtaaaatgca tgacagacag taggggacga
taaactttaa aattctttat agtcttggag 3600 tctttgagat agaaaagaat
atctttttgg ccttatgtca aaagaagtat ggaaaggtga 3660 aagggcggaa
gaaagcagga aaaggaagaa ccatgtatta tatagaggac aatggtgaca 3720
aggtttttct tgaaataatg caaatatgat agattagagg aatttcagta gggaatgctt
3780 ttcacttgaa tttgggtttc ctcttcgatt aagtttggga tcctcatctg
catttgactt 3840 ggagagagaa agaatgaatg ttaggaccta tatctggttt
tctattaact aaagcaagtg 3900 gaaaagactt atttggtatt tttcccacaa
aagtgaaaac ttttctttta ctgtttgtca 3960 aaaaggtgga aatagaaaaa
gccttaatgt attggtgaat acatggttca aagtcatttg 4020 agtagagatg
ttttaaatca ggagtgtcca atcatttggc ttccctggac caccttgaaa 4080
gaattgtctt ggtacacaca taaaatacaa gaacaatagc tgatgagcta aaaaagtcca
4140 tgcataaatc tcatactgtt ttaagaaagt ttatgaattt ctgttagggt
gcattcaaag 4200 ctgtcctggg ccatgtgcgg cctgtgggct gcaggttgga
caagctcctt ataagtaatc 4260 tgtcatagat agttttggag ctgcaaaaca
ggccaaggca taatgggtgg cactcgggat 4320 cccccagatc ccagcctcac
ttcagtctcc ttgctctggt taagaagggg tggtcaactc 4380 tctgcccagc
ttttaaacag cttcattagt gtgaggtgca cctgaaattg atgcctgctg 4440
gtggcctctc agtccagaga gccgtcattt taagctcttt ggcaaatcat acaatactaa
4500 agggatatta ctatgaatgt tttacaaatg cttaaaactc ggtttctgtc
tccatcaacc 4560 taatcttgca atttctaatt tgttcacttt agaaaacatg
gcataaatgc tcaaatactt 4620 ttgcattctt attttcacag cttggagaga
gtggagagat caaagcaatt ggagaactgg 4680 atttgctgtt tatgtctctg
agaaatgcct gcatttgacc agagcaaagc tgaaaaatga 4740 ataactaacc
ccctttccct gctagaaata acaattagat gccccaaagc gattttt 4797 26 20 DNA
Homo sapiens 26 atcagatgga ttactgaatg 20 27 179 PRT Mus musculus 27
Met Ala Val Leu Gln Lys Ser Met Ser Phe Ser Leu Met Gly Thr Leu 1 5
10 15 Ala Ala Ser Cys Leu Leu Leu Ile Ala Leu Trp Ala Gln Glu Ala
Asn 20 25 30 Ala Leu Pro Val Asn Thr Arg Cys Lys Leu Glu Val Ser
Asn Phe Gln 35 40 45 Gln Pro Tyr Ile Val Asn Arg Thr Phe Met Leu
Ala Lys Glu Ala Ser 50 55 60 Leu Ala Asp Asn Asn Thr Asp Val Arg
Leu Ile Gly Glu Lys Leu Phe 65 70 75 80 Arg Gly Val Ser Ala Lys Asp
Gln Cys Tyr Leu Met Lys Gln Val Leu 85 90 95 Asn Phe Thr Leu Glu
Asp Val Leu Leu Pro Gln Ser Asp Arg Phe Gln 100 105 110 Pro Tyr Met
Gln Glu Val Val Pro Phe Leu Thr Lys Leu Ser Asn Gln 115 120 125 Leu
Ser Ser Cys His Ile Ser Gly Asp Asp Gln Asn Ile Gln Lys Asn 130 135
140 Val Arg Arg Leu Lys Glu Thr Val Lys Lys Leu Gly Glu Ser Gly Glu
145 150 155 160 Ile Lys Ala Ile Gly Glu Leu Asp Leu Leu Phe Met Ser
Leu Arg Asn 165 170 175 Ala Cys Val 28 179 PRT Homo sapiens 28 Met
Ala Ala Leu Gln Lys Ser Val Ser Ser Phe Leu Met Gly Thr Leu 1 5 10
15 Ala Thr Ser Cys Leu Leu Leu Leu Ala Leu Leu Val Gln Glu Gly Ala
20 25 30 Ala Ala Pro Ile Ser Ser His Cys Arg Leu Asp Lys Ser Asn
Phe Gln 35 40 45 Gln Pro Tyr Ile Thr Asn Arg Thr Phe Met Leu Ala
Lys Glu Ala Ser 50 55 60 Leu Ala Asp Asn Asn Thr Asp Val Arg Leu
Ile Gly Glu Lys Leu Phe 65 70 75 80 His Gly Val Ser Met Ser Glu Arg
Cys Tyr Leu Met Lys Gln Val Leu 85 90 95 Asn Phe Thr Leu Glu Glu
Ile Leu Phe Pro Gln Ser Asp Arg Phe Arg 100 105 110 Pro Tyr Met Gln
Glu Val Val Pro Phe Leu Ala Arg Leu Ser Asn Arg 115 120 125 Leu Ser
Thr Cys His Ile Glu Gly Asp Asp Leu His Ile Gln Arg Asn 130 135 140
Val Gln Lys Leu Lys Cys Thr Val Lys Lys Leu Gly Glu Ser Gly Glu 145
150 155 160 Ile Lys Ala Ile Gly Glu Leu Asp Leu Leu Phe Met Ser Leu
Arg Asn 165 170 175 Ala Cys Ile 29 5935 DNA Homo sapiens 29
gaattcaagt ccacatgcaa tcaatccgaa tactttgtaa attctcttct tcaaatatcc
60 atctatatag tataagttat tgtaggatca tttaaaaata atgttttgag
acttatgttt 120 gcacaagtaa aatgtcagag agaattagca aatgtatagt
attattttat tttaaaaaat 180 ctatgcttaa aatgtctatt agattgttca
ctactgacat ttccaaactt aacttgacct 240 tggctatgat ttcaaccttt
gtatttgcat ctaccataac tgtgtgctca cttaccatgc 300 tatccgacga
gcatgttccc ctgatgtttt tgccttttgc tctctcgcta acaggctctc 360
ctctcagtta tcaacttttg acacttgtgc gatcggtgat ggctgtcctg cagaaatcta
420 tgagtttttc ccttatgggg actttggccg ccagctgcct gcttctcatt
gccctgtggg 480 cccaggaggc aaatgcgctg cccatcaaca cccggtgcaa
gcttgaggtg tccaacttcc 540 agcagccgta catcgtcaac cgcaccttta
tgctggccaa ggaggtacag ctgcatctct 600 ttctctccat accgccttgc
catttctctg aagcacttgc aaactcttta ggggcgcttt 660 atctccgcag
gtctcactac ctatgttttc tgtctcttta gagactcttt aaggactgga 720
tctttttcta tttctatttc aaggtctcag gaccatttcc tatcttggcc ttcaggacac
780 atatactgaa ttttatctac agaggcgcgt ttagaaagcc acccacgact
gcaatacttt 840 ccatcctgtt gtgctctctt ctgaactcat actctcttgg
ctactcctga gacccactgc 900 ggacatacat ctctacttac aggcttttct
tccatctcct tgtcacccag gcacttaggg 960 ttttctctct ttcaggccag
ccttgcagat aacaacacag acgtccggct catcggggag 1020 aaactgttcc
gaggagtcag tgtaagtcct cactgtgatg agcagggcta gctgcgggag 1080
ctggtggacc ctctgggata gtctgacgta tgacccctgc tgcttcttgt ctacctgcag
1140 gctaaggatc agtgctacct gatgaagcag gtgctcaact tcaccctgga
agacattctg 1200 ctcccccagt cagacaggtt ccggccctac atgcaggagg
tggtgccttt cctgaccaaa 1260 ctcagcaatc agctcagctc ctgtgtaagt
ctggctctgg ctacctatgc tcctctctct 1320 tcctcttcta ttccagtaag
aacccgaggt cctgccctct ctctcttcac aagagtgagg 1380 agggcctcag
caccaccacc atcataggcc acttgaaata ggtcacaaag gctttggctt 1440
caattgagta atactttgag tttgtattag ttaagcttta tttgttttat ccatggaaag
1500 aaatcaactc aaattctgta ggatgagaaa gatgttggga acgaaaaaag
gcctagatag 1560 agaaacagat ctgctgagta cagtacttat gggggggggg
ggcagggggc gatatccact 1620 gagtccaagt acttgttggg agagaaatcc
actgagtaca agtacttgtg ggggaaggaa 1680 tggcacagag caaaagttga
agggaaagag gaagatggag aggcctcaat gttgggggtg 1740 tgaaaggtca
ctcctttttc catgtgatgg agagttaaga aaaatcagtg tgtgagtttg 1800
atgtcttcag acaccccaac tatggcagac tgtgggagac ctggcattta gggaaggcgc
1860 ggcttttcac acgagaaact ttatgctcat ctcttgtgct acactcccac
ctttgatgag 1920 gttaagctca ggtttcgttt ctaccgttct tgctactggt
ggaaacttca gtaggattcc 1980 ccaaagacga ggacagctct tctgtaaggg
agggacctgg atttcagtgt cctagagaac 2040 gaaatagctc agagaatcta
ggtcaacgtg aaatctaggt cacagcgggc aaaaatgact 2100 gaacgcctct
attccaggtg aacggtcacg tgcctcagat atactgaggt attgggctcc 2160
caccggataa gattctgtta gtgagtctgc ttttattttg cagcacatca gtggtgacga
2220 ccagaacatc cagaagaatg tcagaaggct gaaggagaca gtgaaaaagg
tactattggc 2280 aagccacaat actaagccat tcagtaggag acgtggggat
ttctttctct gcttcccagt 2340 ctcttctact ttgtaacatt ttctttgact
tgtctactgt ctggtccatt actcacttag 2400 ctgcacctgc atctagctgg
gtctatagat ctttcaatct gtgtctaaat ttgtaagtca 2460 caattctgga
gctagcagaa agcttagctc agccagtctc atgagcactt gctcggagga 2520
tggcttgtga cagagtcaat gctagaagac agcatccctg attcccagct ctgcacttgc
2580 ctagtggcca cgtgtaatta ctttagcctg attaagtatt tgggaaagcc
aattcccacc 2640 gacctacata atccgaagaa gcatgcattg aaaactagaa
agctgggcac aaacttacta 2700 gagatgattt ttgagctcat taaactgatg
ctctgaaatg tgatcaaatc aacccagaat 2760 aacaacaaaa gagctggatt
tgcaaatagg acaagtattt agaatcactg gtattaacag 2820 ctgtcatctt
aattaaaata tagtgtctat ttagctgcct atttaagatt aaacacaaga 2880
gtggataact tcccaattta ctgggcctgg tttcaataga gtaaaaatat cagtcataga
2940 ttaattatag tgtcatgaaa gtatgagttg gaaacccttt ccttactttt
taccttcatt 3000 tcttagttat tatttttttt tcttcacacc ctgatcaagc
cactagtaag cacctatctg 3060 ctgcgagcta ttatatgact ttacagcaaa
caacattgct gtgtggcctc tttggggaag 3120 ggaacaggat agcaggaggc
tcaggctagc aagtctggac tcaacctaaa gccagaggca 3180 tggttgatag
cagagaaagt gaggctcttc acaagtgggt gtgcttaagt aatcagaaac 3240
aggaaggctc tggttgatgg aattatcagt aagatatcta cccttatctc cttcttctat
3300 agaagctaaa ccgtctctcc ttcttgtgtg taggctgata aacacgcttg
ttttcttttg 3360 agtgttcatg gctttgcaga ttttcagtgc tctgccagtt
cttgttagag ggtttgttac 3420 cttgacacct gggcttggat gttagcatgc
caaaggcaca cacttctgaa tgcctgtgta 3480 aaaggttatt attcatttac
tttgtctttg gaaaggtgaa gtgtgtgtga gaaagaactc 3540 acaggagatg
tattctctgt aggaaaactt ttttttcccc ttaaaagcct ataatccact 3600
ttcagtcaac tttgactttt ataccatgct gtcacatgaa agagtgttta ggcccgctct
3660 cgtggctctg ggaaaagcac caatagggga agaaatgtta tgccgagaaa
tctgactggc 3720 agggaaactg ggtcagagct ccccaaagac cactacaggt
gttaagtagg aacagtcgag 3780 ggtgggttca tataatagaa tggaacagag
ggagggaaga taagctacaa agtttcatag 3840 ggtcctaagt ctttaagata
caaaatagct ggttgggctt cataacaaag gaagtctggg 3900 aaggcagcaa
gcattgagag ggagatggaa agggaaaaaa caatgtagag gatttgaaaa 3960
gctacaaatc ctccacgaga ggatttttct tggaggaatc tagaacaagg gtggtggatt
4020 aggtggatcg cagaaggact tgctttgcca tttgaatctg ggtttttgtc
tctccattga 4080 ggttgagagc gtcacccttt tttaccctgg ataggaggag
gaaagaaggg gtgttttgac 4140 tcctacctgg agttttacta gtttacgcaa
tggaacagac actcgggacc tcctcttgac 4200 aagaaaaaaa aaaaaaaaag
gaaacctgtt gtttctcttg tttgttcttt tgttaagaaa 4260 gcacaggcag
ctgggcatgg tggcccatgc ctttaatccc agcatttggg aggcagaggc 4320
aggtgacttt ctaaattcaa ggccagcctg gtctacaaag tgagttccag gacagccagg
4380 gctatacaga gaaaccctgt ctcgggaaaa aaaaaaaaga agaaaagaaa
agaaaagaag 4440 agaagaggag aggagaggag aggagaggag aggagaggag
aggagaggag aggagaggag 4500 aggagaggag aagagaagag aagagaagag
aagagaagag aagagaagag aagagaagag 4560 aagagaagag aagagaagag
aagagaagag aagagaagag aagagaaaag aaaagagaaa 4620 agaaaagaaa
aaagcaagca agcaagcact ggcaaagcat gcccacatgg gacgtatgtg 4680
ggtctttgag acaaggcttt tgaattgagc gctcatcaat agttgatcat ggtcaggtgg
4740 agggctacct gtcaggccga gccctgctgg cttagcactt aacatctcca
ggtctcagta 4800 tcacttcctg ctgcttagca cagttaggag ttgagcaaac
ctttttttcc aacccccact 4860 aaaatttaat ttacaaaagg cagtgtaatt
tgtgggatac agtgtgataa ttgatctatg 4920 tgtgcattgt gcaaggttca
ataaggtaga tcaataggcc catcaacagc tttatgggtg 4980 tgaaatgcaa
gtaatatagg tagatgcctg tgtgtcctta ggtcagaaag gcatgatttt 5040
aaggtcttgg gcaaatcata ttatactcat gttaaaaatg cattatgttg attatcaatc
5100 ttttagagaa ggctgatact tggttttggt gctcagcaag caaatgtcac
cagctctttc 5160 taactagtac cactttagaa aatgctaccc gtgctcaaat
tggtttgtat tcttattttc 5220 atagcttgga gagagcggag agatcaaagc
gatcggggaa ctggacctgc tgtttatgtc 5280 tctgagaaat gcttgcgtct
gagcgagaag aagctagaaa acgaagaact gctccttcct 5340 gccttctaaa
aagaacaata agatccctga atggactttt ttactaaagg aaagtgagaa 5400
gctaacgtcc accatcatta gaagatttca catgaaacct ggctcagttg aaagagaaaa
5460 tagtgtcaag ttgtccatga gaccagaggt agacttgata accacaaaga
ttcattgaca 5520 atattttatt gtcattgata atgcaacaga aaaagtatgt
actttaaaaa attgtttgaa 5580 aggaggttac ctctcattcc tctagaagaa
aagcctatgt aacttcattt ccataaccaa 5640 tactttatat atgtaagttt
atttattata agtatacatt ttatttatgt cagtttatta 5700 atatggattt
atttatagaa aaattatctg atgttgatat ttgagtataa agcaaataat 5760
atttatgata ataactatag aaacaagata tcttaggctt taataaacac atgaatatca
5820 taaatcttct gtcttgtaat ttttctccct ttaatatcaa caataccatc
atcgtcatca 5880 ttacccaatc attctcatga cttcatgctt gactcatatt
atctggtaaa gtttg 5935
* * * * *