U.S. patent application number 10/284985 was filed with the patent office on 2003-05-22 for human transferases.
This patent application is currently assigned to Incyte Genomics, Inc.. Invention is credited to Bandman, Olga, Corley, Neil C., Gorgone, Gina A., Guegler, Karl J., Hillman, Jennifer L., Lal, Preeti, Patterson, Chandra.
Application Number | 20030095960 10/284985 |
Document ID | / |
Family ID | 22326365 |
Filed Date | 2003-05-22 |
United States Patent
Application |
20030095960 |
Kind Code |
A1 |
Lal, Preeti ; et
al. |
May 22, 2003 |
Human transferases
Abstract
The invention provides three human transferases (HUTRAN) and
polynucleotides which identify and encode HUTRAN. The invention
also provides expression vectors, host cells, antibodies, agonists,
and antagonists. The invention also provides methods for
diagnosing, treating or preventing disorders associated with
expression of HUTRAN.
Inventors: |
Lal, Preeti; (Santa Clara,
CA) ; Bandman, Olga; (Mountain View, CA) ;
Hillman, Jennifer L.; (Santa Cruz, CA) ; Guegler,
Karl J.; (Menlo Park, CA) ; Gorgone, Gina A.;
(Boulder Creek, CA) ; Corley, Neil C.; (Castro
Valley, CA) ; Patterson, Chandra; (San Jose,
CA) |
Correspondence
Address: |
INCYTE GENOMICS, INC.
3160 PORTER DRIVE
PALO ALTO
CA
94304
US
|
Assignee: |
Incyte Genomics, Inc.
Palo Alto
CA
|
Family ID: |
22326365 |
Appl. No.: |
10/284985 |
Filed: |
October 29, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10284985 |
Oct 29, 2002 |
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09490032 |
Jan 21, 2000 |
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6471959 |
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09490032 |
Jan 21, 2000 |
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09109204 |
Jun 30, 1998 |
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6060250 |
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Current U.S.
Class: |
424/94.5 ;
435/193; 435/252.3; 435/320.1; 435/325; 435/6.15; 435/69.1;
536/23.2; 800/8 |
Current CPC
Class: |
A61P 35/00 20180101;
C12N 9/1007 20130101; A61P 37/02 20180101; C12Y 206/01064 20130101;
A61P 1/00 20180101; A61P 29/00 20180101; A61P 43/00 20180101; A61P
25/00 20180101; C12Y 206/01039 20130101; A61P 15/00 20180101; A61K
38/00 20130101; C12N 9/1096 20130101 |
Class at
Publication: |
424/94.5 ;
435/69.1; 435/320.1; 435/325; 435/193; 536/23.2; 435/252.3; 800/8;
435/6 |
International
Class: |
C12Q 001/68; A01K
067/00; C07H 021/04; A61K 038/51; C12N 009/10; C12P 021/02; C12N
005/06 |
Claims
What is claimed is:
1. An isolated polypeptide selected from the group consisting of:
a) a polypeptide comprising an amino acid sequence selected from
the group consisting of SEQ ID NO: 1-3, b) a polypeptide comprising
a naturally occurring amino acid sequence at least 90% identical to
an amino acid sequence selected from the group consisting of SEQ ID
NO: 1-3, c) a biologically active fragment of a polypeptide having
an amino acid sequence selected from the group consisting of SEQ ID
NO: 1-3, and d) an immunogenic fragment of a polypeptide having an
amino acid sequence selected from the group consisting of SEQ ID
NO: 1-3.
2. An isolated polypeptide of claim 1 comprising an amino acid
sequence selected from the group consisting of SEQ ID NO: 1-3.
3. An isolated polynucleotide encoding a polypeptide of claim
1.
4. An isolated polynucleotide encoding a polypeptide of claim
2.
5. An isolated polynucleotide of claim 4 comprising a
polynucleotide sequence selected from the group consisting of SEQ
ID NO:4-6.
6. A recombinant polynucleotide comprising a promoter sequence
operably linked to a polynucleotide of claim 3.
7. A cell transformed with a recombinant polynucleotide of claim
6.
8. A transgenic organism comprising a recombinant polynucleotide of
claim 6.
9. A method of producing a polypeptide of claim 1, the method
comprising: a) culturing a cell under conditions suitable for
expression of the polypeptide, wherein said cell is transformed
with a recombinant polynucleotide, and said recombinant
polynucleotide comprises a promoter sequence operably linked to a
polynucleotide encoding the polypeptide of claim 1, and b)
recovering the polypeptide so expressed.
10. A method of claim 9, wherein the polypeptide comprises an amino
acid sequence selected from the group consisting of SEQ ID NO:
1-3.
11. An isolated antibody which specifically binds to a polypeptide
of claim 1.
12. An isolated polynucleotide selected from the group consisting
of: a) a polynucleotide comprising a polynucleotide sequence
selected from the group consisting of SEQ ID NO:4-6, b) a
polynucleotide comprising a naturally occurring polynucleotide
sequence at least 90% identical to a polynucleotide sequence
selected from the group consisting of SEQ ID NO:4-6, c) a
polynucleotide complementary to a polynucleotide of a), d) a
polynucleotide complementary to a polynucleotide of b), and e) an
RNA equivalent of a)-d).
13. An isolated polynucleotide comprising at least 60 contiguous
nucleotides of a polynucleotide of claim 12.
14. A method of detecting a target polynucleotide in a sample, said
target polynucleotide having a sequence of a polynucleotide of
claim 12, the method comprising: a) hybridizing the sample with a
probe comprising at least 20 contiguous nucleotides comprising a
sequence complementary to said target polynucleotide in the sample,
and which probe specifically hybridizes to said target
polynucleotide, under conditions whereby a hybridization complex is
formed between said probe and said target polynucleotide or
fragments thereof, and b) detecting the presence or absence of said
hybridization complex, and, optionally, if present, the amount
thereof.
15. A method of claim 14, wherein the probe comprises at least 60
contiguous nucleotides.
16. A method of detecting a target polynucleotide in a sample, said
target polynucleotide having a sequence of a polynucleotide of
claim 12, the method comprising: a) amplifying said target
polynucleotide or fragment thereof using polymerase chain reaction
amplification, and b) detecting the presence or absence of said
amplified target polynucleotide or fragment thereof, and,
optionally, if present, the amount thereof.
17. A composition comprising a polypeptide of claim 1 and a
pharmaceutically acceptable excipient.
18. A composition of claim 17, wherein the polypeptide comprises an
amino acid sequence selected from the group consisting of SEQ ID
NO: 1-3.
19. A method for treating a disease or condition associated with
decreased expression of functional HUTRAN, comprising administering
to a patient in need of such treatment the composition of claim
17.
20. A method of screening a compound for effectiveness as an
agonist of a polypeptide of claim 1, the method comprising: a)
exposing a sample comprising a polypeptide of claim 1 to a
compound, and b) detecting agonist activity in the sample.
21. A composition comprising an agonist compound identified by a
method of claim 20 and a pharmaceutically acceptable excipient.
22. A method for treating a disease or condition associated with
decreased expression of functional HUTRAN, comprising administering
to a patient in need of such treatment a composition of claim
21.
23. A method of screening a compound for effectiveness as an
antagonist of a polypeptide of claim 1, the method comprising: a)
exposing a sample comprising a polypeptide of claim 1 to a
compound, and b) detecting antagonist activity in the sample.
24. A composition comprising an antagonist compound identified by a
method of claim 23 and a pharmaceutically acceptable excipient.
25. A method for treating a disease or condition associated with
overexpression of functional HUTRAN, comprising administering to a
patient in need of such treatment a composition of claim 24.
26. A method of screening for a compound that specifically binds to
the polypeptide of claim 1, the method comprising: a) combining the
polypeptide of claim 1 with at least one test compound under
suitable conditions, and b) detecting binding of the polypeptide of
claim 1 to the test compound, thereby identifying a compound that
specifically binds to the polypeptide of claim 1.
27. A method of screening for a compound that modulates the
activity of the polypeptide of claim 1, the method comprising: a)
combining the polypeptide of claim 1 with at least one test
compound under conditions permissive for the activity of the
polypeptide of claim 1, b) assessing the activity of the
polypeptide of claim 1 in the presence of the test compound, and c)
comparing the activity of the polypeptide of claim 1 in the
presence of the test compound with the activity of the polypeptide
of claim 1 in the absence of the test compound, wherein a change in
the activity of the polypeptide of claim 1 in the presence of the
test compound is indicative of a compound that modulates the
activity of the polypeptide of claim 1.
28. A method of screening a compound for effectiveness in altering
expression of a target polynucleotide, wherein said target
polynucleotide comprises a sequence of claim 5, the method
comprising: a) exposing a sample comprising the target
polynucleotide to a compound, under conditions suitable for the
expression of the target polynucleotide, b) detecting altered
expression of the target polynucleotide, and c) comparing the
expression of the target polynucleotide in the presence of varying
amounts of the compound and in the absence of the compound.
29. A method of assessing toxicity of a test compound, the method
comprising: a) treating a biological sample containing nucleic
acids with the test compound, b) hybridizing the nucleic acids of
the treated biological sample with a probe comprising at least 20
contiguous nucleotides of a polynucleotide of claim 12 under
conditions whereby a specific hybridization complex is formed
between said probe and a target polynucleotide in the biological
sample, said target polynucleotide comprising a polynucleotide
sequence of a polynucleotide of claim 12 or fragment thereof, c)
quantifying the amount of hybridization complex, and d) comparing
the amount of hybridization complex in the treated biological
sample with the amount of hybridization complex in an untreated
biological sample, wherein a difference in the amount of
hybridization complex in the treated biological sample is
indicative of toxicity of the test compound.
30. A method for a diagnostic test for a condition or disease
associated with the expression of HUTRAN in a biological sample,
the method comprising: a) combining the biological sample with an
antibody of claim 11, under conditions suitable for the antibody to
bind the polypeptide and form an antibody:polypeptide complex, and
b) detecting the complex, wherein the presence of the complex
correlates with the presence of the polypeptide in the biological
sample.
31. The antibody of claim 11, wherein the antibody is: a) a
chimeric antibody, b) a single chain antibody, c) a Fab fragment,
d) a F(ab').sub.2 fragment, or e) a humanized antibody.
32. A composition comprising an antibody of claim 11 and an
acceptable excipient.
33. A method of diagnosing a condition or disease associated with
the expression of HUTRAN in a subject, comprising administering to
said subject an effective amount of the composition of claim
32.
34. A composition of claim 32, wherein the antibody is labeled.
35. A method of diagnosing a condition or disease associated with
the expression of HUTRAN in a subject, comprising administering to
said subject an effective amount of the composition of claim
34.
36. A method of preparing a polyclonal antibody with the
specificity of the antibody of claim 11, the method comprising: a)
immunizing an animal with a polypeptide consisting of an amino acid
sequence selected from the group consisting of SEQ ID NO:1-3, or an
immunogenic fragment thereof, under conditions to elicit an
antibody response, b) isolating antibodies from the animal, and c)
screening the isolated antibodies with the polypeptide, thereby
identifying a polyclonal antibody which specifically binds to a
polypeptide comprising an amino acid sequence selected from the
group consisting of SEQ ID NO:1-3.
37. A polyclonal antibody produced by a method of claim 36.
38. A composition comprising the polyclonal antibody of claim 37
and a suitable carrier.
39. A method of making a monoclonal antibody with the specificity
of the antibody of claim 11, the method comprising: a) immunizing
an animal with a polypeptide consisting of an amino acid sequence
selected from the group consisting of SEQ ID NO:1-3, or an
immunogenic fragment thereof, under conditions to elicit an
antibody response, b) isolating antibody producing cells from the
animal, c) fusing the antibody producing cells with immortalized
cells to form monoclonal antibody-producing hybridoma cells, d)
culturing the hybridoma cells, and e) isolating from the culture
monoclonal antibody which specifically binds to a polypeptide
comprising an amino acid sequence selected from the group
consisting of SEQ ID NO: 1-3.
40. A monoclonal antibody produced by a method of claim 39.
41. A composition comprising the monoclonal antibody of claim 40
and a suitable carrier.
42. The antibody of claim 11, wherein the antibody is produced by
screening a Fab expression library.
43. The antibody of claim 11, wherein the antibody is produced by
screening a recombinant immunoglobulin library.
44. A method of detecting a polypeptide comprising an amino acid
sequence selected from the group consisting of SEQ ID NO: 1-3 in a
sample, the method comprising: a) incubating the antibody of claim
11 with the sample under conditions to allow specific binding of
the antibody and the polypeptide, and b) detecting specific
binding, wherein specific binding indicates the presence of a
polypeptide comprising an amino acid sequence selected from the
group consisting of SEQ ID NO: 1-3 in the sample.
45. A method of purifying a polypeptide comprising an amino acid
sequence selected from the group consisting of SEQ ID NO:1-3 from a
sample, the method comprising: a) incubating the antibody of claim
11 with the sample under conditions to allow specific binding of
the antibody and the polypeptide, and b) separating the antibody
from the sample and obtaining the purified polypeptide comprising
an amino acid sequence selected from the group consisting of SEQ ID
NO: 1-3.
46. A microarray wherein at least one element of the microarray is
a polynucleotide of claim 13.
47. A method of generating an expression profile of a sample which
contains polynucleotides, the method comprising: a) labeling the
polynucleotides of the sample, b) contacting the elements of the
microarray of claim 46 with the labeled polynucleotides of the
sample under conditions suitable for the formation of a
hybridization complex, and c) quantifying the expression of the
polynucleotides in the sample.
48. An array comprising different nucleotide molecules affixed in
distinct physical locations on a solid substrate, wherein at least
one of said nucleotide molecules comprises a first oligonucleotide
or polynucleotide sequence specifically hybridizable with at least
30 contiguous nucleotides of a target polynucleotide, and wherein
said target polynucleotide is a polynucleotide of claim 12.
49. An array of claim 48, wherein said first oligonucleotide or
polynucleotide sequence is completely complementary to at least 30
contiguous nucleotides of said target polynucleotide.
50. An array of claim 48, wherein said first oligonucleotide or
polynucleotide sequence is completely complementary to at least 60
contiguous nucleotides of said target polynucleotide.
51. An array of claim 48, wherein said first oligonucleotide or
polynucleotide sequence is completely complementary to said target
polynucleotide.
52. An array of claim 48, which is a microarray.
53. An array of claim 48, further comprising said target
polynucleotide hybridized to a nucleotide molecule comprising said
first oligonucleotide or polynucleotide sequence.
54. An array of claim 48, wherein a linker joins at least one of
said nucleotide molecules to said solid substrate.
55. An array of claim 48, wherein each distinct physical location
on the substrate contains multiple nucleotide molecules, and the
multiple nucleotide molecules at any single distinct physical
location have the same sequence, and each distinct physical
location on the substrate contains nucleotide molecules having a
sequence which differs from the sequence of nucleotide molecules at
another distinct physical location on the substrate.
56. A polypeptide of claim 1, comprising the amino acid sequence of
SEQ ID NO:1.
57. A polypeptide of claim 1, comprising the amino acid sequence of
SEQ ID NO:2.
58. A polypeptide of claim 1, comprising the amino acid sequence of
SEQ ID NO:3.
63. A polynucleotide of claim 12, comprising the polynucleotide
sequence of SEQ ID NO:4.
64. A polynucleotide of claim 12, comprising the polynucleotide
sequence of SEQ ID NO:5.
65. A polynucleotide of claim 12, comprising the polynucleotide
sequence of SEQ ID NO:6.
Description
[0001] This application is a divisional application of U.S.
application Ser. No. 09/490,032, filed Jan. 21, 2000, which is a
divisional application of U.S. application Ser. No. 09/109,204,
filed Jun. 30, 1998, now U.S. Pat. No. 6,060,250, issued May 9,
2000, all of which are entitled HUMAN TRANSFERASES, and all of
which are expressly incorporated herein by reference.
FIELD OF THE INVENTION
[0002] This invention relates to nucleic acid and amino acid
sequences of human transferases and to the use of these sequences
in the diagnosis, treatment, and prevention of
autoimmune/inflammatory, neurological, reproductive, and
gastrointestinal disorders and cancer.
BACKGROUND OF THE INVENTION
[0003] Transferases, enzymes that catalyze group transfer
reactions, are classified by the type of group transferred.
One-carbon groups are transferred; for example, methyltransferases
transfer methyl groups from S-adenosyl-methionine to substrates.
Nitrogenous groups are transferred; for example, aminotransferases
transfer amino groups. Other groups transferred include aldehyde or
ketone, acyl, glycosyl, alkyl and aryl other than methyl,
phosphorus-containing, sulfur-containing, and selenium-containing
groups.
[0004] The enzyme glutamine-phenylpyruvate aminotransferase, also
known as glutamine transaminase K (GTK), catalyzes several
reactions with a pyridoxal phosphate cofactor. GTK catalyzes the
reversible conversion of L-glutamine and phenylpyruvate to
2-oxoglutaramate and L-phenylalanine. L-methionine, L-histidine,
and L-tyrosine can substitute for L-glutamine in this reaction. GTK
also catalyzes the conversion of kynurenine to kynurenic acid.
Kynurenic acid, a tryptophan metabolite, is an antagonist of the
N-methyl-D-aspartate (NMDA) receptor in the brain and may exert a
neuromodulatory function. Alteration of the kynurenine metabolic
pathway may be among the causative factors leading to several
neurological disorders. GTK also possesses cysteine conjugate
.beta.-lyase activity which is involved in the metabolism of
halogenated xenobiotics conjugated to glutathione. GTK action on
the cysteine conjugates of xenobiotics yields metabolites that are
nephrotoxic in rats and neurotoxic in humans. The neurotoxicity may
be related to the kynurenine aminotransferase activity of GTK. GTK
is expressed in kidney, liver, and brain. Both cytosolic and
mitochondrial forms exist. Human and rat GTK genes have been
isolated which encode proteins of 422 and 423 amino acids
respectively. Both human and rat GTKs contain a putative pyridoxal
phosphate binding site. (ExPASy ENZYME: EC 2.6.1.64; Perry, S. J.
et al. (1993) Mol. Pharmacol. 43:660-665; Perry, S. et al. (1995)
FEBS Lett. 360:277-280; and Alberati-Giani, D. et al. (1995) J.
Neurochem. 64:1448-1455.)
[0005] The enzyme kynurenine/.alpha.-aminoadipate aminotransferase
(AadAT) catalyzes two reactions with a pyridoxal phosphate
cofactor. AadAT catalyzes the reversible conversion of
.alpha.-aminoadipate and .alpha.-ketoglutarate to
.alpha.-ketoadipate and L-glutamate. This conversion is involved in
lysine metabolism. AadAT also catalyzes the transamination of
kynurenine acid to kynurenic acid. As described above, kynurenic
acid is an NMDA receptor antagonist. Both soluble and mitochondrial
forms of AadAT have been purified. A soluble AadAT is expressed in
rat kidney, liver, and brain. The rat AadAT nucleotide gene encodes
a protein of 425 amino acids which contains a putative pyridoxal
phosphate binding site. (Nakatani, Y. et al. (1970) Biochim.
Biophys. Acta 198:219-228; Buchli, R. et al. (1995) J. Biol. Chem.
270:29330-29335.)
[0006] Protein-arginine methyltransferases catalyze the
posttranslational methylation of arginine residues in proteins,
resulting in the mono- and dimethylation of arginine on the
guanidino group. Known substrates are histones, heterogeneous
nuclear ribonucleoproteins (hnRNPs), and myelin basic protein. This
otherwise unusual posttranslational modification is common in
hnRNPs and may regulate their function. hnRNPs function in the
nucleus in mRNA processing, splicing, and transport into the
cytoplasm. Homologous protein-arginine methyltransferases that
methylate hnRNPs have been cloned from yeast, rat, and man. These
protein-arginine methyltransferases contain five sequence motifs,
termed region I, post-region I, region II, region III, and
post-region III, that may be involved in binding
S-adenosyl-methionine. One human gene (HRMT1L1) encodes a 433 amino
acid protein. The other human gene (HRMT1L2) may be alternatively
spliced to yield three protein-arginine methyltransferases, of
length 343, 347, and 361 amino acids respectively, with different
amino termini. The protein encoded by the cloned rat
protein-arginine methyltransferase gene (PRMT1) interacts with the
TIS21 protein and the homologous BTG1 protein. The
intermediate-early TIS21 protein is the product of a gene induced
by treatment of cells with mitogens such as epidermal growth
factor, and the BTG1 protein is the product of a human gene located
near a chromosome translocation breakpoint associated with chronic
lymphocytic leukemia. The HRMT1L2 protein interacts with the
cytoplasmic domain of the interferon receptor. This interaction
suggests that protein methylation may be an important signaling
mechanism for cytokine receptors. (Lin, W. -J. et al. (1996) J.
Biol. Chem. 271:15034-15044; Abramovich, C. et al. (1997) EMBO J.
16:260-266; and Scott, H. S. et al. (1998) Genomics
48:330-340.)
[0007] The discovery of new human transferases and the
polynucleotides encoding them satisfies a need in the art by
providing new compositions which are useful in the diagnosis,
treatment, and prevention of autoimmune/inflammatory, neurological,
reproductive, and gastrointestinal disorders and cancer.
SUMMARY OF THE INVENTION
[0008] The invention features substantially purified polypeptides,
human transferases, referred to collectively as "HUTRAN" and
individually as "HUTRAN-1", "HUTRAN-2", and "HUTRAN-3." In one
aspect, the invention provides a substantially purified polypeptide
comprising an amino acid sequence selected from the group
consisting of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3 (SEQ ID
NO:1-3) and fragments thereof.
[0009] The invention further provides a substantially purified
variant having at least 90% amino acid identity to the amino acid
sequences of SEQ ID NO: 1-3 or to fragments of any of these
sequences. The invention also provides an isolated and purified
polynucleotide encoding the polypeptide comprising an amino acid
sequence selected from the group consisting of SEQ ID NO: 1-3 and
fragments thereof. The invention also includes an isolated and
purified polynucleotide variant having at least 70% polynucleotide
sequence identity to the polynucleotide encoding the polypeptide
comprising an amino acid sequence selected from the group
consisting of SEQ ID NO: 1-3 and fragments thereof.
[0010] Additionally, the invention provides an isolated and
purified polynucleotide which hybridizes under stringent conditions
to the polynucleotide encoding the polypeptide comprising an amino
acid sequence selected from the group consisting of SEQ ID NO: 1-3
and fragments thereof, as well as an isolated and purified
polynucleotide having a sequence which is complementary to the
polynucleotide encoding the polypeptide comprising the amino acid
sequence selected from the group consisting of SEQ ID NO: 1-3 and
fragments thereof.
[0011] The invention also provides an isolated and purified
polynucleotide comprising a polynucleotide sequence selected from
the group consisting of SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6
(SEQ ID NO:4-6), and fragments thereof. The invention further
provides an isolated and purified polynucleotide variant having at
least 70% polynucleotide sequence identity to the polynucleotide
sequence comprising a polynucleotide sequence selected from the
group consisting of SEQ ID NO:4-6 and fragments thereof, as well as
an isolated and purified polynucleotide having a sequence which is
complementary to the polynucleotide comprising a polynucleotide
sequence selected from the group consisting of SEQ ID NO:4-6 and
fragments thereof.
[0012] The invention further provides an expression vector
containing at least a fragment of the polynucleotide encoding the
polypeptide comprising an amino acid sequence selected from the
group consisting of SEQ ID NO: 1-3 and fragments thereof. In
another aspect, the expression vector is contained within a host
cell.
[0013] The invention also provides a method for producing a
polypeptide comprising the amino acid sequence selected from the
group consisting of SEQ ID NO:1-3 and fragments thereof, the method
comprising the steps of: (a) culturing the host cell containing an
expression vector containing at least a fragment of a
polynucleotide encoding the polypeptide under conditions suitable
for the expression of the polypeptide; and (b) recovering the
polypeptide from the host cell culture.
[0014] The invention also provides a pharmaceutical composition
comprising a substantially purified polypeptide having the amino
acid sequence selected from the group consisting of SEQ ID NO: 1-3
and fragments thereof in conjunction with a suitable pharmaceutical
carrier.
[0015] The invention further includes a purified antibody which
binds to a polypeptide comprising the amino acid sequence selected
from the group consisting of SEQ ID NO:1-3 and fragments thereof,
as well as a purified agonist and a purified antagonist to the
polypeptide. The invention also provides a method for treating or
preventing an autoimmune/inflammatory disorder, the method
comprising administering to a subject in need of such treatment an
effective amount of an antagonist of the polypeptide having an
amino acid sequence selected from the group consisting of SEQ ID
NO: 1-3 and fragments thereof.
[0016] The invention also provides a method for treating or
preventing a neurological disorder, the method comprising
administering to a subject in need of such treatment an effective
amount of a pharmaceutical composition comprising a substantially
purified polypeptide having an amino acid sequence selected from
the group consisting of SEQ ID NO: 1-3 and fragments thereof.
[0017] The invention also provides a method for treating or
preventing a reproductive disorder, the method comprising
administering to a subject in need of such treatment an effective
amount of a pharmaceutical composition comprising a substantially
purified polypeptide having an amino acid sequence selected from
the group consisting of SEQ ID NO: 1-3 and fragments thereof.
[0018] The invention also provides a method for treating or
preventing a gastrointestinal disorder, the method comprising
administering to a subject in need of such treatment an effective
amount of a pharmaceutical composition comprising a substantially
purified polypeptide having an amino acid sequence selected from
the group consisting of SEQ ID NO: 1-3 and fragments thereof.
[0019] The invention also provides a method for treating or
preventing a cancer, the method comprising administering to a
subject in need of such treatment an effective amount of an
antagonist of the polypeptide having an amino acid sequence
selected from the group consisting of SEQ ID NO: 1-3 and fragments
thereof.
[0020] The invention also provides a method for detecting a
polynucleotide encoding the polypeptide comprising the amino acid
sequence selected from the group consisting of SEQ ID NO: 1-3 and
fragments thereof in a, biological sample containing nucleic acids,
the method comprising the steps of: (a) hybridizing the complement
of the polynucleotide sequence encoding the polypeptide comprising
the amino acid sequence selected from the group consisting of SEQ
ID NO:1-3 and fragments thereof to at least one of the nucleic
acids of the biological sample, thereby forming a hybridization
complex; and (b) detecting the hybridization complex, wherein the
presence of the hybridization complex correlates with the presence
of a polynucleotide encoding the polypeptide in the biological
sample. In one aspect, the method further comprises amplifying the
polynucleotide prior to the hybridizing step.
BRIEF DESCRIPTION OF THE FIGURES AND TABLES
[0021] FIGS. 1A and 1B show the amino acid sequence alignment
between HUTRAN-1 (1815528; SEQ ID NO:1) and human
glutamine-phenylpyruvate aminotransferase (GI 758591; SEQ ID
NO:30), produced using the multisequence alignment program of
LASERGENE software (DNASTAR Inc, Madison Wis.).
[0022] FIGS. 2A and 2B show the amino acid sequence alignment
between HUTRAN-2 (2150892; SEQ ID NO:2) and rat
kynurenine/.alpha.-aminoadipate aminotransferase (GI 1050752; SEQ
ID NO:31).
[0023] FIGS. 3A, 3B, and 3C show the amino acid sequence alignment
between HUTRAN-3 (2525071; SEQ ID NO:3) and human arginine
methyltransferase (GI 1808648; SEQ ID NO:32).
[0024] In Table 1, columns 1 and 2 show the sequence identification
numbers (SEQ ID NO:) of the amino acid and nucleic acid sequence,
respectively. Column 3 shows the Clone ID of the Incyte Clone in
which nucleic acids encoding each HUTRAN were first identified, and
column 4, the cDNA library of this clone. Column 5 shows the Incyte
clones (and libraries) and shotgun sequences useful as fragments in
hybridization technologies, and which are part of the consensus
nucleotide sequence of each HUTRAN.
[0025] Table 2 shows various properties of the polypeptides of the
invention: column 1 references the SEQ ID NO; column 2 shows the
number of amino acid residues; column 3, potential phosphorylation
sites; column 4, potential glycosylation sites; column 5, signature
sequences associated with known proteins; column 6, the identity of
the protein; and column 7, analytical methods used to identify the
protein through sequence homologies, protein motifs, and protein
signatures.
[0026] Table 3 shows the tissue expression of each nucleic acid
sequence by northern analysis, diseases or conditions associated
with this tissue expression, and the vector into which each cDNA
was cloned.
[0027] Table 4 describes the tissues used in cDNA library
construction.
[0028] Table 5 describes the programs, algorithms, databases, and
qualifying scores used to analyze HUTRAN. The first column of Table
5 shows the tool, program, or algorithm; the second column, the
database; the third column, a brief description; and the fourth
column (where applicable), scores for determining the strength of a
match between two sequences (the higher the value, the more
homologous).
DESCRIPTION OF THE INVENTION
[0029] 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.
[0030] It must be noted that as used herein and in the appended
claims, the singular forms "a," "an," and "the" include plural
reference unless the context clearly dictates otherwise. Thus, for
example, a reference to "a host cell" includes a plurality of such
host cells, and a reference to "an antibody" is a reference to one
or more antibodies and equivalents thereof known to those skilled
in the art, and so forth.
[0031] 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 cited for the
purpose of describing and disclosing the cell lines, vectors, and
methodologies which are reported in the publications and 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 by virtue of prior invention.
[0032] Definitions
[0033] "HUTRAN," as used herein, refers to the amino acid sequences
of substantially purified HUTRAN obtained from any species,
particularly a mammalian species, including bovine, ovine, porcine,
murine, equine, and preferably the human species, from any source,
whether natural, synthetic, semi-synthetic, or recombinant.
[0034] The term "agonist," as used herein, refers to a molecule
which, when bound to HUTRAN, increases or prolongs the duration of
the effect of HUTRAN. Agonists may include proteins, nucleic acids,
carbohydrates, or any other molecules which bind to and modulate
the effect of HUTRAN.
[0035] An "allelic variant," as this term is used herein, is an
alternative form of the gene encoding HUTRAN. Allelic variants may
result from at least one mutation in the nucleic acid sequence and
may result in altered mRNAs or in polypeptides whose structure or
function may or may not be altered. Any given natural or
recombinant gene may have none, one, or many allelic forms. Common
mutational changes which give rise to allelic variants 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.
[0036] "Altered" nucleic acid sequences encoding HUTRAN, as
described herein, include those sequences with deletions,
insertions, or substitutions of different nucleotides, resulting in
a polynucleotide the same as HUTRAN or a polypeptide with at least
one functional characteristic of HUTRAN. Included within this
definition are polymorphisms which may or may not be readily
detectable using a particular oligonucleotide probe of the
polynucleotide encoding HUTRAN, and improper or unexpected
hybridization to allelic variants, with a locus other than the
normal chromosomal locus for the polynucleotide sequence encoding
HUTRAN. The encoded protein may also be "altered," and may contain
deletions, insertions, or substitutions of amino acid residues
which produce a silent change and result in a functionally
equivalent HUTRAN. 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 or immunological activity
of HUTRAN 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; and phenylalanine
and tyrosine.
[0037] The terms "amino acid" or "amino acid sequence," as used
herein, refer to an oligopeptide, peptide, polypeptide, or protein
sequence, or a fragment of any of these, and to naturally occurring
or synthetic molecules. In this context, "fragments," "immunogenic
fragments," or "antigenic fragments" refer to fragments of HUTRAN
which are preferably at least 5 to about 15 amino acids in length,
most preferably at least 14 amino acids, and which retain some
biological activity or immunological activity of HUTRAN. Where
"amino acid sequence" is recited herein to refer to an amino acid
sequence of a naturally occurring protein molecule, "amino acid
sequence" and like terms are not meant to limit the amino acid
sequence to the complete native amino acid sequence associated with
the recited protein molecule.
[0038] "Amplification," as used herein, relates to the production
of additional copies of a nucleic acid sequence. Amplification is
generally carried out using polymerase chain reaction (PCR)
technologies well known in the art. (See, e.g., Dieffenbach, C. W.
and G. S. Dveksler (1995) PCR Primer, a Laboratory Manual, Cold
Spring Harbor Press, Plainview, N.Y., pp. 1-5.)
[0039] The term "antagonist," as it is used herein, refers to a
molecule which, when bound to HUTRAN, decreases the amount or the
duration of the effect of the biological or immunological activity
of HUTRAN. Antagonists may include proteins, nucleic acids,
carbohydrates, antibodies, or any other molecules which decrease
the effect of HUTRAN.
[0040] As used herein, the term "antibody" refers to intact
molecules as well as to fragments thereof, such as Fab,
F(ab').sub.2, and Fv fragments, which are capable of binding the
epitopic determinant. Antibodies that bind HUTRAN polypeptides can
be prepared using intact polypeptides or using fragments containing
small peptides of interest as the immunizing antigen. The
polypeptide or oligopeptide used to immunize an animal (e.g., a
mouse, a rat, or a rabbit) can be derived from the translation of
RNA, or synthesized chemically, and can be conjugated to a carrier
protein if desired. Commonly used carriers that are chemically
coupled to peptides include bovine serum albumin, thyroglobulin,
and keyhole limpet hemocyanin (KLH). The coupled peptide is then
used to immunize the animal.
[0041] The term "antigenic determinant," as used herein, refers to
that fragment of a molecule (i.e., an epitope) that makes contact
with a particular antibody. When a protein or a fragment of a
protein is used to immunize a host animal, numerous regions of the
protein may induce the production of antibodies which bind
specifically to antigenic determinants (given regions or
three-dimensional structures on the protein). An antigenic
determinant may compete with the intact antigen (i.e., the
immunogen used to elicit the immune response) for binding to an
antibody.
[0042] The term "antisense," as used herein, refers to any
composition containing a nucleic acid sequence which is
complementary to the "sense" strand of a specific nucleic acid
sequence. Antisense molecules may be produced by any method
including synthesis or transcription. Once introduced into a cell,
the complementary nucleotides combine with natural sequences
produced by the cell to form duplexes and to block either
transcription or translation. The designation "negative" can refer
to the antisense strand, and the designation "positive" can refer
to the sense strand.
[0043] As used herein, the term "biologically active," refers to a
protein having structural, regulatory, or to biochemical functions
of a naturally occurring molecule. Likewise, "immunologically
active" refers to the capability of the natural, recombinant, or
synthetic HUTRAN, or of any oligopeptide thereof, to induce a
specific immune response in appropriate animals or cells and to
bind with specific antibodies.
[0044] The terms "complementary" or "complementarity," as used
herein, refer to the natural binding of polynucleotides by base
pairing. For example, the sequence "5' A-G-T 3'" binds to the
complementary sequence "3' T-C-A 5'." Complementarity between two
single-stranded molecules may be "partial," such that only some of
the nucleic acids bind, or it may be "complete," such that total
complementarity exists between the single stranded molecules. The
degree of complementarity between nucleic acid strands has
significant effects on the efficiency and strength of the
hybridization between the nucleic acid strands. This is of
particular importance in amplification reactions, which depend upon
binding between nucleic acids strands, and in the design and use of
peptide nucleic acid (PNA) molecules.
[0045] A "composition comprising a given polynucleotide sequence"
or a "composition comprising a given amino acid sequence," as these
terms are used herein, refer broadly to any composition containing
the given polynucleotide or amino acid sequence. The composition
may comprise a dry formulation or an aqueous solution. Compositions
comprising polynucleotide sequences encoding HUTRAN or fragments of
HUTRAN may be employed as hybridization probes. The probes may be
stored in freeze-dried form and may be associated with a
stabilizing agent such as a carbohydrate. In hybridizations, the
probe may be deployed in an aqueous solution containing salts,
e.g., NaCl, detergents, e.g., sodium dodecyl sulfate (SDS), and
other components, e.g., Denhardt's solution, dry milk, salmon sperm
DNA, etc.
[0046] "Consensus sequence," as used herein, refers to a nucleic
acid sequence which has been resequenced to resolve uncalled bases,
extended using XL-PCR (Perkin Elmer, Norwalk, Conn.) in the 5'
and/or the 3' direction, and resequenced, or which has been
assembled from the overlapping sequences of more than one Incyte
Clone using a computer program for fragment assembly, such as the
GELVIEW fragment assembly system (GCG, Madison, Wis.). Some
sequences have been both extended and assembled to produce the
consensus sequence.
[0047] As used herein, the term "correlates with expression of a
polynucleotide" indicates that the detection of the presence of
nucleic acids, the same or related to a nucleic acid sequence
encoding HUTRAN, by Northern analysis is indicative of the presence
of nucleic acids encoding HUTRAN in a sample, and thereby
correlates with expression of the transcript from the
polynucleotide encoding HUTRAN.
[0048] A "deletion," as the term is used herein, refers to a change
in the amino acid or nucleotide sequence that results in the
absence of one or more amino acid residues or nucleotides.
[0049] The term "derivative," as used herein, refers to the
chemical modification of a polypeptide sequence, or a
polynucleotide sequence. Chemical modifications of a polynucleotide
sequence can include, for example, replacement of hydrogen by an
alkyl, acyl, or amino group. A derivative polynucleotide encodes a
polypeptide which retains at least one biological or immunological
function of the natural molecule. A derivative polypeptide is one
modified by glycosylation, pegylation, or any similar process that
retains at least one biological or immunological function of the
polypeptide from which it was derived.
[0050] The term "similarity," as used herein, refers to a degree of
complementarity. There may be partial similarity or complete
similarity. The word "identity" may substitute for the word
"similarity." A partially complementary sequence that at least
partially inhibits an identical sequence from hybridizing to a
target nucleic acid is referred to as "substantially similar." The
inhibition of hybridization of the completely complementary
sequence to the target sequence may be examined using a
hybridization assay (Southern or Northern blot, solution
hybridization, and the like) under conditions of reduced
stringency. A substantially similar sequence or hybridization probe
will compete for and inhibit the binding of a completely similar
(identical) sequence to the target sequence under conditions of
reduced stringency. This is not to say that conditions of reduced
stringency are such that non-specific binding is permitted, as
reduced stringency conditions require that the binding of two
sequences to one another be a specific (i.e., a selective)
interaction. The absence of non-specific binding may be tested by
the use of a second target sequence which lacks even a partial
degree of complementarity (e.g., less than about 30% similarity or
identity). In the absence of non-specific binding, the
substantially similar sequence or probe will not hybridize to the
second non-complementary target sequence.
[0051] The phrases "percent identity" or "% identity" refer to the
percentage of sequence similarity found in a comparison of two or
more amino acid or nucleic acid sequences. Percent identity can be
determined electronically, e.g., by using the MEGALIGN program
(DNASTAR, Inc., Madison Wis.). The MEGALIGN program can create
alignments between two or more sequences according to different
methods, e.g., the clustal method. (See, e.g., Higgins, D. G. and
P. M. Sharp (1988) Gene 73:237-244.) The clustal algorithm groups
sequences into clusters by examining the distances between all
pairs. The clusters are aligned pairwise and then in groups. The
percentage similarity between two amino acid sequences, e.g.,
sequence A and sequence B, is calculated by dividing the length of
sequence A, minus the number of gap residues in sequence A, minus
the number of gap residues in sequence B, into the sum of the
residue matches between sequence A and sequence B, times one
hundred. Gaps of low or of no similarity between the two amino acid
sequences are not included in determining percentage similarity.
Percent identity between nucleic acid sequences can also be counted
or calculated by other methods known in the art, e.g., the Jotun
Hein method. (See, e.g., Hein, J. (1990) Methods Enzymol.
183:626-645.) Identity between sequences can also be determined by
other methods known in the art, e.g., by varying hybridization
conditions.
[0052] "Human artificial chromosomes" (HACs), as described herein,
are linear microchromosomes which may contain DNA sequences of
about 6 kb to 10 Mb in size, and which contain all of the elements
required for stable mitotic chromosome segregation and maintenance.
(See, e.g., Harrington, J. J. et al. (1997) Nat Genet.
15:345-355.)
[0053] The term "humanized antibody," as used herein, refers to
antibody molecules in which the amino acid sequence in the
non-antigen binding regions has been altered so that the antibody
more closely resembles a human antibody, and still retains its
original binding ability.
[0054] "Hybridization," as the term is used herein, refers to any
process by which a strand of nucleic acid binds with a
complementary strand through base pairing.
[0055] As used herein, the term "hybridization complex" refers to a
complex formed between two nucleic acid sequences by virtue of the
formation of hydrogen bonds between complementary bases. A
hybridization complex may be formed in solution (e.g., C.sub.0t or
R.sub.0t analysis) or formed between one nucleic acid sequence
present in solution and another nucleic acid sequence immobilized
on a solid support (e.g., paper, membranes, filters, chips, pins or
glass slides, or any other appropriate substrate to which cells or
their nucleic acids have been fixed).
[0056] The words "insertion" or "addition," as used herein, refer
to changes in an amino acid or nucleotide sequence resulting in the
addition of one or more amino acid residues or nucleotides,
respectively, to the sequence found in the naturally occurring
molecule.
[0057] "Immune response" can refer to conditions associated with
inflammation, trauma, immune disorders, or infectious or genetic
disease, etc. These conditions can be characterized by expression
of various factors, e.g., cytokines, chemokines, and other
signaling molecules, which may affect cellular and systemic defense
systems.
[0058] The term "microarray," as used herein, refers to an
arrangement of distinct polynucleotides arrayed on a substrate,
e.g., paper, nylon or any other type of membrane, filter, chip,
glass slide, or any other suitable solid support.
[0059] The terms "element" or "array element" as used herein in a
microarray context, refer to hybridizable polynucleotides arranged
on the surface of a substrate.
[0060] The term "modulate," as it appears herein, refers to a
change in the activity of HUTRAN. For example, modulation may cause
an increase or a decrease in protein activity, binding
characteristics, or any other biological, functional, or
immunological properties of HUTRAN.
[0061] The phrases "nucleic acid" or "nucleic acid sequence," as
used herein, refer to a nucleotide, oligonucleotide,
polynucleotide, or any fragment thereof. These phrases also refer
to DNA or RNA of genomic or synthetic origin which may be
single-stranded or double-stranded and may represent the sense or
the antisense strand, to peptide nucleic acid (PNA), or to any
DNA-like or RNA-like material. In this context, "fragments" refers
to those nucleic acid sequences which, when translated, would
produce polypeptides retaining some functional characteristic,
e.g., antigenicity, or structural domain characteristic, e.g.,
ATP-binding site, of the full-length polypeptide.
[0062] The terms "operably associated" or "operably linked," as
used herein, refer to functionally related nucleic acid sequences.
A promoter is operably associated or operably linked with a coding
sequence if the promoter controls the translation of the encoded
polypeptide. While operably associated or operably linked nucleic
acid sequences can be contiguous and in the same reading frame,
certain genetic elements, e.g., repressor genes, are not
contiguously linked to the sequence encoding the polypeptide but
still bind to operator sequences that control expression of the
polypeptide.
[0063] The term "oligonucleotide," as used herein, refers to a
nucleic acid sequence of at least about 6 nucleotides to 60
nucleotides, preferably about 15 to 30 nucleotides, and most
preferably about 20 to 25 nucleotides, which can be used in PCR
amplification or in a hybridization assay or microarray. As used
herein, the term "oligonucleotide" is substantially equivalent to
the terms "amplimer," "primer," "oligomer," and "probe," as these
terms are commonly defined in the art.
[0064] "Peptide nucleic acid" (PNA), as used herein, refers to an
antisense molecule or anti-gene agent which comprises an
oligonucleotide of at least about 5 nucleotides in length linked to
a peptide backbone of amino acid residues ending in lysine. The
terminal lysine confers solubility to the composition. PNAs
preferentially bind complementary single stranded DNA or RNA and
stop transcript elongation, and may be pegylated to extend their
lifespan in the cell. (See, e.g., Nielsen, P. E. et al. (1993)
Anticancer Drug Des. 8:53-63.)
[0065] The term "sample," as used herein, is used in its broadest
sense. A biological sample suspected of containing nucleic acids
encoding HUTRAN, or fragments thereof, or HUTRAN itself, may
comprise a bodily fluid; an extract from a cell, chromosome,
organelle, or membrane isolated from a cell; a cell; genomic DNA,
RNA, or cDNA, in solution or bound to a solid support; a tissue; a
tissue print; etc.
[0066] As used herein, the terms "specific binding" or
"specifically binding" refer to that interaction between a protein
or peptide and an agonist, an antibody, or an antagonist. The
interaction is dependent upon the presence of a particular
structure of the protein, e.g., the antigenic determinant or
epitope, recognized by the binding molecule. For example, if an
antibody is specific for epitope "A," the presence of a polypeptide
containing the epitope A, or the presence of free unlabeled A, in a
reaction containing free labeled A and the antibody will reduce the
amount of labeled A that binds to the antibody.
[0067] As used herein, the term "stringent conditions" refers to
conditions which permit hybridization between polynucleotides and
the claimed polynucleotides. Stringent conditions can be defined by
salt concentration, the concentration of organic solvent, e.g.,
formamide, temperature, and other conditions well known in the art.
In particular, stringency can be increased by reducing the
concentration of salt, increasing the concentration of formamide,
or raising the hybridization temperature.
[0068] The term "substantially purified," as used herein, refers to
nucleic acid or amino acid sequences that are removed from their
natural environment and are isolated or separated, and are at least
about 60% free, preferably about 75% free, and most preferably
about 90% free from other components with which they are naturally
associated.
[0069] A "substitution," as used herein, refers to the replacement
of one or more amino acids or nucleotides by different amino acids
or nucleotides, respectively.
[0070] "Transformation," as defined herein, describes a process by
which exogenous DNA enters and changes a recipient cell.
Transformation may occur under natural or artificial conditions
according to various methods well known in the art, and may rely on
any known method for the insertion of foreign nucleic acid
sequences into a prokaryotic or eukaryotic host cell. The method
for transformation is selected based on the type of host cell being
transformed and may include, but is not limited to, viral
infection, electroporation, heat shock, lipofection, and particle
bombardment. The term "transformed" cells includes stably
transformed cells in which the inserted DNA is capable of
replication either as an autonomously replicating plasmid or as
part of the host chromosome, as well as transiently transformed
cells which express the inserted DNA or RNA for limited periods of
time.
[0071] A "variant" of HUTRAN polypeptides, as used herein, refers
to an amino acid sequence that is altered by one or more amino acid
residues. The variant may have "conservative" changes, wherein a
substituted amino acid has similar structural or chemical
properties (e.g., replacement of leucine with isoleucine). More
rarely, a variant may have "nonconservative" changes (e.g.,
replacement of glycine with tryptophan). Analogous minor variations
may also include amino acid deletions or insertions, or both.
Guidance in determining which amino acid residues may be
substituted, inserted, or deleted without abolishing biological or
immunological activity may be found using computer programs well
known in the art, for example, LASERGENE software.
[0072] The term "variant," when used in the context of a
polynucleotide sequence, may encompass a polynucleotide sequence
related to HUTRAN. This definition may also include, for example,
"allelic" (as defined above), "splice," "species," or "polymorphic"
variants. A splice variant may have significant identity to a
reference molecule, but will generally have a greater or lesser
number of polynucleotides due to alternate splicing of exons during
mRNA processing. The corresponding polypeptide may possess
additional functional domains or an absence of domains. Species
variants are polynucleotide sequences that vary from one species to
another. The resulting polypeptides generally will have significant
amino acid identity relative to each other. A polymorphic variant
is a variation in the polynucleotide sequence of a particular gene
between individuals of a given species. Polymorphic variants also
may encompass "single nucleotide polymorphisms" (SNPs) in which the
polynucleotide sequence varies by one base. The presence of SNPs
may be indicative of, for example, a certain population, a disease
state, or a propensity for a disease state.
[0073] The Invention
[0074] The invention is based on the discovery of three new human
transferases (HUTRAN), the polynucleotides encoding HUTRAN, and the
use of these compositions for the diagnosis, treatment, or
prevention of autoimmune/inflammatory, neurological, reproductive,
and gastrointestinal disorders and cancer. Table 1 summarizes the
sequence identification numbers, identifying clone numbers, and
libraries of HUTRAN.
[0075] As shown in Table 2, each HUTRAN has been characterized with
regard to its chemical and structural similarity with transferase
molecules. As shown in FIGS. 1A and 1B, HUTRAN-1 and human
glutamine-phenylpyruvate aminotransferase (GI 758591; SEQ ID NO:30)
share 49% identity. As shown in FIGS. 2A and 2B, HUTRAN-2 and rat
kynurenine/.alpha.-aminoadipate aminotransferase (GI 1050752; SEQ
ID NO:31) share 71% identity. As shown in FIGS. 3A, 3B, and 3C,
HUTRAN-3 and human arginine methyltransferase (GI 1808648; SEQ ID
NO:32) share 27% identity.
[0076] In Table 3, northern analysis shows the expression of HUTRAN
sequences in various libraries, of which at least 42% are
immortalized or cancerous, at least 18% are in fetal or
proliferating tissue, at least 9% involve trauma, and at least 14%
involve immune response. Of particular note is the expression of
HUTRAN in male and female reproductive, nervous, and
gastrointestinal tissues.
[0077] A preferred HUTRAN variant is one which has at least about
80%, more preferably at least about 90%, and most preferably at
least about 95% amino acid sequence identity to the HUTRAN amino
acid sequence, and which contains at least one functional or
structural characteristic of HUTRAN.
[0078] The invention also encompasses polynucleotides which encode
HUTRAN. In a particular embodiment, the invention encompasses a
polynucleotide sequence comprising the sequence of SEQ ID NO:4,
which encodes a HUTRAN. In a further embodiment, the invention
encompasses the polynucleotide sequence comprising the sequence of
SEQ ID NO:5. In a further embodiment, the invention encompasses the
polynucleotide sequence comprising the sequence of SEQ ID NO:6.
[0079] The invention also encompasses a variant of a polynucleotide
sequence encoding HUTRAN. In particular, such a variant
polynucleotide sequence will have at least about 70%, more
preferably at least about 85%, and most preferably at least about
95% polynucleotide sequence identity to the polynucleotide sequence
encoding HUTRAN. Any one of the polynucleotide variants described
above can encode an amino acid sequence which contains at least one
functional or structural characteristic of HUTRAN.
[0080] It will be appreciated by those skilled in the art that as a
result of the degeneracy of the genetic code, a multitude of
polynucleotide sequences encoding HUTRAN, some bearing minimal
similarity to the polynucleotide sequences of any known and
naturally occurring gene, may be produced. Thus, the invention
contemplates each and every possible variation of polynucleotide
sequence that could be made by selecting combinations based on
possible codon choices. These combinations are made in accordance
with the standard triplet genetic code as applied to the
polynucleotide sequence of naturally occurring HUTRAN, and all such
variations are to be considered as being specifically
disclosed.
[0081] Although nucleotide sequences which encode HUTRAN and its
variants are preferably capable of hybridizing to the nucleotide
sequence of the naturally occurring HUTRAN under appropriately
selected conditions of stringency, it may be advantageous to
produce nucleotide sequences encoding HUTRAN or its derivatives
possessing a substantially different codon usage, e.g., inclusion
of non-naturally occurring codons. 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 utilized by the host.
Other reasons for substantially altering the nucleotide sequence
encoding HUTRAN 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 sequence.
[0082] The invention also encompasses production of DNA sequences
which encode HUTRAN and HUTRAN derivatives, or fragments thereof,
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 well known in the art.
Moreover, synthetic chemistry may be used to introduce mutations
into a sequence encoding HUTRAN or any fragment thereof.
[0083] Also encompassed by the invention are polynucleotide
sequences that are capable of hybridizing to the claimed
polynucleotide sequences, and, in particular, to those shown in SEQ
ID NO:4-6 and fragments thereof under various conditions of
stringency. (See, e.g., Wahl, G. M. and S. L. Berger (1987) Methods
Enzymol. 152:399-407; Kimmel, A. R. (1987) Methods Enzymol.
152:507-511.) For example, stringent salt concentration will
ordinarily be less than about 750 mM NaCl and 75 mM trisodium
citrate, preferably less than about 500 mM NaCl and 50 mM trisodium
citrate, and most preferably less than about 250 mM NaCl and 25 mM
trisodium citrate. Low stringency hybridization can be obtained in
the absence of organic solvent, e.g., formamide, while high
stringency hybridization can be obtained in the presence of at
least about 35% formamide, and most preferably at least about 50%
formamide. Stringent temperature conditions will ordinarily include
temperatures of at least about 30.degree. C., more preferably of at
least about 37.degree. C., and most preferably of at least about
42.degree. C. Varying additional parameters, such as hybridization
time, the concentration of detergent, e.g., sodium dodecyl sulfate
(SDS), and the inclusion or exclusion of carrier DNA, are well
known to those skilled in the art. Various levels of stringency are
accomplished by combining these various conditions as needed. In a
preferred embodiment, hybridization will occur at 30.degree. C. in
750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In a more
preferred embodiment, hybridization will occur at 37.degree. C. in
500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and
100 .mu.g/ml denatured salmon sperm DNA (ssDNA). In a most
preferred embodiment, hybridization will occur at 42.degree. C. in
250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and
200 .mu.g/ml ssDNA. Useful variations on these conditions will be
readily apparent to those skilled in the art.
[0084] The washing steps which follow hybridization can also vary
in stringency. Wash stringency conditions can be defined by salt
concentration and by temperature. As above, wash stringency can be
increased by decreasing salt concentration or by increasing
temperature. For example, stringent salt concentration for the wash
steps will preferably be less than about 30 mM NaCl and 3 mM
trisodium citrate, and most preferably less than about 15 mM NaCl
and 1.5 mM trisodium citrate. Stringent temperature conditions for
the wash steps will ordinarily include temperature of at least
about 25.degree. C., more preferably of at least about 42.degree.
C., and most preferably of at least about 68.degree. C. In a
preferred embodiment, wash steps will occur at 25.degree. C. in 30
mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In a more preferred
embodiment, wash steps will occur at 42.degree. C. in 15 mM NaCl,
1.5 mM trisodium citrate, and 0.1% SDS. In a most preferred
embodiment, wash steps will occur at 68.degree. C. in 15 mM NaCl,
1.5 mM trisodium citrate, and 0.1% SDS. Additional variations on
these conditions will be readily apparent to those skilled in the
art.
[0085] 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 (US
Biochemical Corp, Cleveland, Ohio), Taq polymerase (Perkin Elmer),
thermostable T7 polymerase (Amersham, Chicago, Ill.), or
combinations of recombinant polymerases and proofreading
exonucleases such as the ELONGASE amplification system marketed by
Gibco BRL (Gaithersburg, Md.). Preferably, the process is automated
with machines such as the MICROLAB 2200 liquid transfer system
(Hamilton, Reno, Nev.), PTC200 thermal cycler (MJ Research,
Watertown, Mass.) and the ABI 377 DNA sequencers (Perkin
Elmer).
[0086] The nucleic acid sequences encoding HUTRAN may be extended
utilizing a partial nucleotide sequence and employing various
PCR-based 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 and
nested primers to amplify unknown sequence from genomic DNA within
a cloning vector. (See, e.g., Sarkar, G. (1993) PCR Methods Applic.
2:318-322.) Another method, inverse PCR, uses primers that extend
in divergent directions to amplify unknown sequence from a
circularized template. The template is derived from restriction
fragments comprising a known genomic locus and surrounding
sequences. (See, e.g., Triglia, T. et al. (1988) Nucleic Acids Res.
16:8186.) A third method, capture PCR, involves PCR amplification
of DNA fragments adjacent to known sequences in human and yeast
artificial chromosome DNA. (See, e.g., Lagerstrom, M. et al. (1991)
PCR Methods Applic. 1:111-119.) In this method, multiple
restriction enzyme digestions and ligations may be used to insert
an engineered double-stranded sequence into a region of unknown
sequence before performing PCR. Other methods which may be used to
retrieve unknown sequences are known in the art. (See, e.g.,
Parker, J. D. et al. (1991) Nucleic Acids Res. 19:3055-306).
Additionally, one may use PCR, nested primers, and PROMOTERFINDER
libraries to walk genomic DNA (Clontech, Palo Alto, Calif.). This
procedure avoids the need to screen libraries and is useful in
finding intron/exon junctions. For all PCR-based methods, primers
may be designed using commercially available software, such as
OLIGO 4.06 primer analysis software (National Biosciences Inc.,
Plymouth, Minn.) or another appropriate program, to be about 22 to
30 nucleotides in length, to have a GC content of about 50% or
more, and to anneal to the template at temperatures of about
68.degree. C. to 72.degree. C.
[0087] When screening for full-length cDNAs, it is preferable to
use libraries that have been size-selected to include larger cDNAs.
In addition, random-primed libraries, which often include sequences
containing the 5' regions of genes, are 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 5'
non-transcribed regulatory regions.
[0088] Capillary electrophoresis systems which are commercially
available may be used to analyze 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 nucleotide-specific, laser-stimulated
fluorescent dyes, and a charge coupled device camera for detection
of the emitted wavelengths. 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 sequencing small DNA fragments which may be present
in limited amounts in a particular sample.
[0089] In another embodiment of the invention, polynucleotide
sequences or fragments thereof which encode HUTRAN may be cloned in
recombinant DNA molecules that direct expression of HUTRAN, or
fragments or functional equivalents thereof, 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 used to express
HUTRAN.
[0090] The nucleotide sequences of the present invention can be
engineered using methods generally known in the art in order to
alter HUTRAN-encoding sequences for a variety of purposes
including, but not limited to, modification of 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, oligonucleotide-mediated site-directed
mutagenesis may be used to introduce mutations that create new
restriction sites, alter glycosylation patterns, change codon
preference, produce splice variants, and so forth.
[0091] In another embodiment, sequences encoding HUTRAN may be
synthesized, in whole or in part, using chemical methods well known
in the art. (See, e.g., Caruthers, M. H. et al. (1980) Nucl. Acids
Res. Symp. Ser. 215-223, and Horn, T. et al. (1980) Nucl. Acids
Res. Symp. Ser. 225-232.) Alternatively, HUTRAN itself or a
fragment thereof may be synthesized using chemical methods. For
example, peptide synthesis can be performed using various
solid-phase techniques. (See, e.g., Roberge, J. Y. et al. (1995)
Science 269:202-204.) Automated synthesis may be achieved using the
ABI 431A peptide synthesizer (Perkin Elmer). Additionally, the
amino acid sequence of HUTRAN, or any part thereof, may be altered
during direct synthesis and/or combined with sequences from other
proteins, or any part thereof, to produce a variant
polypeptide.
[0092] The peptide may be substantially purified by preparative
high performance liquid chromatography. (See, e.g, Chiez, R. M. and
F. Z. Regnier (1990) Methods Enzymol. 182:392-421.) The composition
of the synthetic peptides may be confirmed by amino acid analysis
or by sequencing. (See, e.g., Creighton, T. (1984) Proteins,
Structures and Molecular Properties, W H Freeman and Co., New York,
N.Y.)
[0093] In order to express a biologically active HUTRAN, the
nucleotide sequences encoding HUTRAN or derivatives thereof may be
inserted into an appropriate expression vector, i.e., a vector
which contains the necessary elements for transcriptional and
translational control of the inserted coding sequence in a suitable
host. These elements include regulatory sequences, such as
enhancers, constitutive and inducible promoters, and 5' and 3'
untranslated regions in the vector and in polynucleotide sequences
encoding HUTRAN. Such elements may vary in their strength and
specificity. Specific initiation signals may also be used to
achieve more efficient translation of sequences encoding HUTRAN.
Such signals include the ATG initiation codon and adjacent
sequences, e.g. the Kozak sequence. In cases where sequences
encoding HUTRAN and its initiation codon and upstream regulatory
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 fragment
thereof, is inserted, exogenous translational control signals
including an in-frame ATG initiation codon should be provided by
the vector. 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 appropriate for the particular host cell system used.
(See, e.g., Scharf, D. et al. (1994) Results Probl. Cell Differ.
20:125-162.)
[0094] Methods which are well known to those skilled in the art may
be used to construct expression vectors containing sequences
encoding HUTRAN and appropriate transcriptional and translational
control elements. These methods include in vitro recombinant DNA
techniques, synthetic techniques, and in vivo genetic
recombination. (See, e.g., Sambrook, J. et al. (1989) Molecular
Cloning, A Laboratory Manual, Cold Spring Harbor Press, Plainview,
N.Y., ch. 4, 8, and 16-17; and Ausubel, F. M. et al. (1995, and
periodic supplements) Current Protocols in Molecular Biology, John
Wiley & Sons, New York, N.Y., ch. 9, 13, and 16.)
[0095] A variety of expression vector/host systems may be utilized
to contain and express sequences encoding HUTRAN. These include,
but are not limited to, microorganisms such as bacteria transformed
with recombinant bacteriophage, plasmid, or cosmid DNA expression
vectors; yeast transformed with yeast expression vectors; insect
cell systems infected with viral expression vectors (e.g.,
baculovirus); plant cell systems transformed with viral expression
vectors (e.g., cauliflower mosaic virus (CaMV) or tobacco mosaic
virus (TMV)) or with bacterial expression vectors (e.g., Ti or
pBR322 plasmids); or animal cell systems. The invention is not
limited by the host cell employed.
[0096] In bacterial systems, a number of cloning and expression
vectors may be selected depending upon the use intended for
polynucleotide sequences encoding HUTRAN. For example, routine
cloning, subcloning, and propagation of polynucleotide sequences
encoding HUTRAN can be achieved using a multifunctional E. coli
vector such as BLUESCRIPT (Stratagene) or PSport1 plasmid (Life
Technologies, Inc., Gaithersburg, Md.). Ligation of sequences
encoding HUTRAN into the vector's multiple cloning site disrupts
the lacZ gene, allowing a colorimetric screening procedure for
identification of transformed bacteria containing recombinant
molecules. In addition, these vectors may be useful for in vitro
transcription, dideoxy sequencing, single strand rescue with helper
phage, and creation of nested deletions in the cloned sequence.
(See, e.g., Van Heeke, G. and S. M. Schuster (1989) J. Biol. Chem.
264:5503-5509.) When large quantities of HUTRAN are needed, e.g.
for the production of antibodies, vectors which direct high level
expression of HUTRAN may be used. For example, vectors containing
the strong, inducible T5 or T7 bacteriophage promoter may be
used.
[0097] Yeast expression systems may be used for production of
HUTRAN. A number of vectors containing constitutive or inducible
promoters, such as alpha factor, alcohol oxidase, and PGH, may be
used in the yeast Saccharomyces cerevisiae or Pichia pastoris. In
addition, such vectors direct either the secretion or intracellular
retention of expressed proteins and enable integration of foreign
sequences into the host genome for stable propagation. (See, e.g.,
Ausubel, supra; and Grant et al. (1987) Methods Enzymol.
153:516-54; Scorer, C. A. et al. (1994) Bio/Technology
12:181-184.)
[0098] Plant systems may also be used for expression of HUTRAN.
Transcription of sequences encoding HUTRAN may be driven viral
promoters, e.g., the 35S and 19S promoters of CaMV 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.
(See, e.g., 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. (See, e.g., Hobbs, S. or Murry, L.
E. in McGraw Hill Yearbook of Science and Technology (1992) McGraw
Hill, New York, N.Y.; pp. 191-196.)
[0099] In mammalian cells, a number of viral-based expression
systems may be utilized. In cases where an adenovirus is used as an
expression vector, sequences encoding HUTRAN 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 infective virus which expresses HUTRAN in host cells. (See,
e.g., Logan, J. and T. Shenk (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. SV40 or EBV-based vectors may
also be used for high-level protein expression.
[0100] Human artificial chromosomes (HACs) may also be employed to
deliver larger fragments of DNA than can be contained in and
expressed from a plasmid. HACs of about 6 kb to 10 Mb are
constructed and delivered via conventional delivery methods
(liposomes, polycationic amino polymers, or vesicles) for
therapeutic purposes.
[0101] For long term production of recombinant proteins in
mammalian systems, stable expression of HUTRAN in cell lines is
preferred. For example, sequences encoding HUTRAN can be
transformed into cell lines 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 about 1 to 2 days in enriched media before
being switched to selective media. The purpose of the selectable
marker is to confer resistance to a selective agent, and its
presence allows growth and recovery of cells which successfully
express the introduced sequences. Resistant clones of stably
transformed cells may be propagated using tissue culture techniques
appropriate to the cell type.
[0102] 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 and adenine
phosphoribosyltransferase genes, for use in tk.sup.- or apr.sup.-
cells, respectively. (See, e.g., Wigler, M. et al. (1977) Cell
11:223-232; and Lowy, I. et al. (1980) Cell 22:817-823.) Also,
antimetabolite, antibiotic, or herbicide resistance can be used as
the basis for selection. For example, dhfr confers resistance to
methotrexate; neo confers resistance to the aminoglycosides
neomycin and G-418; and als or pat confer resistance to
chlorsulfuron and phosphinotricin acetyltransferase, respectively.
(See, e.g., Wigler, M. et al. (1980) Proc. Natl. Acad. Sci.
77:3567-3570; Colbere-Garapin, F. et al (1981) J. Mol. Biol.
150:1-14; and Murry, supra.) Additional selectable genes have been
described, e.g., trpB and hisD, which alter cellular requirements
for metabolites. (See, e.g., Hartman, S. C. and R. C. Mulligan
(1988) Proc. Natl. Acad. Sci. 85:8047-8051.) Visible markers, e.g.,
anthocyanins, green fluorescent proteins (GFP) (Clontech, Palo
Alto, Calif.), .beta.-glucuronidase and its substrate
.beta.-D-glucuronoside, or luciferase and its substrate luciferin
may be used. These markers can be used not only to identify
transformants, but also to quantify the amount of transient or
stable protein expression attributable to a specific vector system.
(See, e.g., Rhodes, C. A. et al. (1995) Methods Mol. Biol.
55:121-131.)
[0103] Although the presence/absence of marker gene expression
suggests that the gene of interest is also present, the presence
and expression of the gene may need to be confirmed. For example,
if the sequence encoding HUTRAN is inserted within a marker gene
sequence, transformed cells containing sequences encoding HUTRAN
can be identified by the absence of marker gene function.
Alternatively, a marker gene can be placed in tandem with a
sequence encoding HUTRAN 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.
[0104] In general, host cells that contain the nucleic acid
sequence encoding HUTRAN and that express HUTRAN 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 hybridizations, PCR amplification, 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 sequences.
[0105] Immunological methods for detecting and measuring the
expression of HUTRAN using either specific polyclonal or monoclonal
antibodies are known in the art. Examples of such techniques
include enzyme-linked immunosorbent assays (ELISAs),
radioimmunoassays (RIAs), and fluorescence activated cell sorting
(FACS). A two-site, monoclonal-based immunoassay utilizing
monoclonal antibodies reactive to two non-interfering epitopes on
HUTRAN is preferred, but a competitive binding assay may be
employed. These and other assays are well known in the art. (See,
e.g., Hampton, R. et al. (1990) Serological Methods, a Laboratory
Manual, APS Press, St Paul, Minn., Section IV; Coligan, J. E. et
al. (1997 and periodic supplements) Current Protocols in
Immunology, Greene Pub. Associates and Wiley-Interscience, New
York, N.Y.; and Maddox, D. E. et al. (1983) J. Exp. Med.
158:1211-1216).
[0106] 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 labeled
hybridization or PCR probes for detecting sequences related to
polynucleotides encoding HUTRAN include oligolabeling, nick
translation, end-labeling, or PCR amplification using a labeled
nucleotide. Alternatively, the sequences encoding HUTRAN, or any
fragments 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 synthesize RNA probes in
vitro by addition of an appropriate RNA polymerase such as T7, T3,
or SP6 and labeled nucleotides. These procedures may be conducted
using a variety of commercially available kits, such as those
provided by Amersham Pharmacia Biotech (Piscataway, N.J.), Promega
(Madison, Wis.), and U.S. Biochemical Corp. (Cleveland, Ohio).
Suitable reporter molecules or labels which may be used for ease of
detection include radionuclides, enzymes, fluorescent,
chemiluminescent, or chromogenic agents, as well as substrates,
cofactors, inhibitors, magnetic particles, and the like.
[0107] Host cells transformed with nucleotide sequences encoding
HUTRAN may be cultured under conditions suitable for the expression
and recovery of the protein from cell culture. The protein produced
by a transformed cell may be secreted or retained 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 HUTRAN may be designed to
contain signal sequences which direct secretion of HUTRAN through a
prokaryotic or eukaryotic cell membrane.
[0108] In addition, a host cell strain may be chosen for its
ability to modulate 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 specify
protein targeting, folding, and/or activity. Different host cells
which have specific cellular machinery and characteristic
mechanisms for post-translational activities (e.g., CHO, HeLa,
MDCK, HEK293, and WI38), are available from the American Type
Culture Collection (ATCC, Manassas, Va.) and may be chosen to
ensure the correct modification and processing of the foreign
protein.
[0109] In another embodiment of the invention, natural, modified,
or recombinant nucleic acid sequences encoding HUTRAN may be
ligated to a heterologous sequence resulting in translation of a
fusion protein in any of the aforementioned host systems. For
example, a chimeric HUTRAN protein containing a heterologous moiety
that can be recognized by a commercially available antibody may
facilitate the screening of peptide libraries for inhibitors of
HUTRAN activity. Heterologous protein and peptide moieties may also
facilitate purification of fusion proteins using commercially
available affinity matrices. Such moieties include, but are not
limited to, glutathione S-transferase (GST), maltose binding
protein (MBP), thioredoxin (Trx), calmodulin binding peptide (CBP),
6-His, FLAG, c-myc, and hemagglutinin (HA). GST, MBP, Trx, CBP, and
6-His enable purification of their cognate fusion proteins on
immobilized glutathione, maltose, phenylarsine oxide, calmodulin,
and metal-chelate resins, respectively. FLAG, c-myc, and
hemagglutinin (HA) enable immunoaffinity purification of fusion
proteins using commercially available monoclonal and polyclonal
antibodies that specifically recognize these epitope tags. A fusion
protein may also be engineered to contain a proteolytic cleavage
site located between the HUTRAN encoding sequence and the
heterologous protein sequence, so that HUTRAN may be cleaved away
from the heterologous moiety following purification. Methods for
fusion protein expression and purification are discussed in
Ausubel, F. M. et al. (1995 and periodic supplements) Current
Protocols in Molecular Biology, John Wiley & Sons, New York,
N.Y., ch 10. A variety of commercially available kits may also be
used to facilitate expression and purification of fusion
proteins.
[0110] In a further embodiment of the invention, synthesis of
radiolabeled HUTRAN may be achieved in vitro using the TNT rabbit
reticulocyte lysate or wheat germ extract systems (Promega,
Madison, Wis.). These systems couple transcription and translation
of protein-coding sequences operably associated with the T7, T3, or
SP6 promoters. Translation takes place in the presence of a
radiolabeled amino acid precursor, preferably
.sup.35S-methionine.
[0111] Fragments of HUTRAN may be produced not only by recombinant
production, but also by direct peptide synthesis using solid-phase
techniques. (See, e.g., Creighton, supra pp. 55-60.) Protein
synthesis may be performed by manual techniques or by automation.
Automated synthesis may be achieved, for example, using the Applied
Biosystems 431A peptide synthesizer (Perkin Elmer). Various
fragments of HUTRAN may be synthesized separately and then combined
to produce the full length molecule.
[0112] Therapeutics
[0113] Chemical and structural similarity, e.g., in the context of
sequences and motifs, exists between HUTRAN-1 and
glutamine-phenylpyruvat- e aminotransferase from man (GI 758591),
between HUTRAN-2 and kynurenine/.alpha.-aminoadipate
aminotransferase from rat (GI 1050752), and between HUTRAN-3 and
arginine methyltransferase from man (GI 1808648). In addition,
HUTRAN is expressed in cancerous, inflamed, male and female
reproductive, nervous, and gastrointestinal tissues. Therefore,
HUTRAN appears to play a role in autoimmune/inflammatory,
neurological, reproductive, and gastrointestinal disorders, and
cancer.
[0114] Therefore, in one embodiment, an antagonist of HUTRAN may be
administered to a subject to treat or prevent an
autoimmune/inflammatory disorder. Such an autoimmune/inflammatory
disorder may include, but is not limited to, acquired
immunodeficiency syndrome (AIDS), Addison's disease, adult
respiratory distress syndrome, allergies, ankylosing spondylitis,
amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolytic
anemia, autoimmune thyroiditis, bronchitis, cholecystitis, contact
dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis,
diabetes mellitus, emphysema, episodic lymphopenia with
lymphocytotoxins, erythroblastosis fetalis, erythema nodosum,
atrophic gastritis, glomerulonephritis, Goodpasture's syndrome,
gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophilia,
irritable bowel syndrome, multiple sclerosis, myasthenia gravis,
myocardial or pericardial inflammation, osteoarthritis,
osteoporosis, pancreatitis, polymyositis, psoriasis, Reiter's
syndrome, rheumatoid arthritis, scleroderma, Sjogren's syndrome,
systemic anaphylaxis, systemic lupus erythematosus, systemic
sclerosis, thrombocytopenic purpura, ulcerative colitis, uveitis,
Werner syndrome, complications of cancer, hemodialysis, and
extracorporeal circulation, viral, bacterial, fungal, parasitic,
protozoal, and helminthic infections, and trauma. In one aspect, an
antibody which specifically binds HUTRAN 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
HUTRAN.
[0115] In an additional embodiment, a vector expressing the
complement of the polynucleotide encoding HUTRAN may be
administered to a subject to treat or prevent an
autoimmune/inflammatory disorder including, but not limited to,
those described above.
[0116] In another embodiment, HUTRAN or a fragment or derivative
thereof may be administered to a subject to treat or prevent a
neurological disorder. Such neurological disorders can include, but
are not limited to, epilepsy, ischemic cerebrovascular disease,
stroke, cerebral neoplasms, Alzheimer's disease, Pick's disease,
Huntington's disease, dementia, Parkinson's disease and other
extrapyramidal disorders, amyotrophic lateral sclerosis and other
motor neuron disorders, progressive neural muscular atrophy,
retinitis pigmentosa, hereditary ataxias, multiple sclerosis and
other demyelinating diseases, bacterial and viral meningitis, brain
abscess, subdural empyema, epidural abscess, suppurative
intracranial thrombophlebitis, myelitis and radiculitis, viral
central nervous system disease; prion diseases including kuru,
Creutzfeldt-Jakob disease, and Gerstmann-Straussler-Scheinker
syndrome; fatal familial insomnia, nutritional and metabolic
diseases of the nervous system, neurofibromatosis, tuberous
sclerosis, cerebelloretinal hemangioblastomatosis,
encephalotrigeminal syndrome, mental retardation and other
developmental disorders of the central nervous system, cerebral
palsy, neuroskeletal disorders, autonomic nervous system disorders,
cranial nerve disorders, spinal cord diseases, muscular dystrophy
and other neuromuscular disorders, peripheral nervous system
disorders, dermatomyositis and polymyositis; inherited, metabolic,
endocrine, and toxic myopathies; myasthenia gravis, periodic
paralysis; mental disorders including mood, anxiety, and
schizophrenic disorders; akathesia, amnesia, catatonia, diabetic
neuropathy, tardive dyskinesia, dystonias, paranoid psychoses,
postherpetic neuralgia, and Tourette's disorder.
[0117] In another embodiment, a vector capable of expressing HUTRAN
or a fragment or derivative thereof may be administered to a
subject to treat or prevent a neurological disorder including, but
not limited to, those described above.
[0118] In a further embodiment, a pharmaceutical composition
comprising a substantially purified HUTRAN in conjunction with a
suitable pharmaceutical carrier may be administered to a subject to
treat or prevent a neurological disorder including, but not limited
to, those provided above.
[0119] In still another embodiment, an agonist which modulates the
activity of HUTRAN may be administered to a subject to treat or
prevent a neurological disorder including, but not limited to,
those listed above.
[0120] In another embodiment, HUTRAN or a fragment or derivative
thereof may be administered to a subject to treat or prevent a
reproductive disorder. Such reproductive disorders can include, but
are not limited to, disorders of prolactin production; infertility,
including tubal disease, ovulatory defects, and endometriosis;
disruptions of the estrous cycle, disruptions of the menstrual
cycle, polycystic ovary syndrome, ovarian hyperstimulation
syndrome, endometrial and ovarian tumors, uterine fibroids,
autoimmune disorders, ectopic pregnancies, and teratogenesis;
cancer of the breast, fibrocystic breast disease, and galactorrhea;
disruptions of spermatogenesis, abnormal sperm physiology, cancer
of the testis, cancer of the prostate, benign prostatic
hyperplasia, prostatitis, Peyronie's disease, carcinoma of the male
breast, and gynecomastia.
[0121] In another embodiment, a vector capable of expressing HUTRAN
or a fragment or derivative thereof may be administered to a
subject to treat or prevent a reproductive disorder including, but
not limited to, those described above.
[0122] In a further embodiment, a pharmaceutical composition
comprising a substantially purified HUTRAN in conjunction with a
suitable pharmaceutical carrier may be administered to a subject to
treat or prevent a reproductive disorder including, but not limited
to, those provided above.
[0123] In still another embodiment, an agonist which modulates the
activity of HUTRAN may be administered to a subject to treat or
prevent a reproductive disorder including, but not limited to,
those listed above.
[0124] In another embodiment, HUTRAN or a fragment or derivative
thereof may be administered to a subject to treat or prevent a
gastrointestinal disorder. Such gastrointestinal disorders can
include, but are not limited to, dysphagia, peptic esophagitis,
esophageal spasm, esophageal stricture, esophageal carcinoma,
dyspepsia, indigestion, gastritis, gastric carcinoma, anorexia,
nausea, emesis, gastroparesis, antrat or pyloric edema, abdominal
angina, pyrosis, gastroenteritis, intestinal obstruction,
infections of the intestinal tract, peptic ulcer, cholelithiasis,
cholecystitis, cholestasis, pancreatitis, pancreatic carcinoma,
biliary tract disease, hepatitis, hyperbilirubinemia, cirrhosis,
passive congestion of the liver, hepatoma, infectious colitis,
ulcerative colitis, ulcerative proctitis, Crohn's disease,
Whipple's disease, Mallory-Weiss syndrome, colonic carcinoma,
colonic obstruction, irritable bowel syndrome, short bowel
syndrome, diarrhea, constipation, gastrointestinal hemorrhage, and
acquired immunodeficiency syndrome (AIDS) enteropathy.
[0125] In another embodiment, a vector capable of expressing HUTRAN
or a fragment or derivative thereof may be administered to a
subject to treat or prevent a gastrointestinal disorder including,
but not limited to, those described above.
[0126] In a further embodiment, a pharmaceutical composition
comprising a substantially purified HUTRAN in conjunction with a
suitable pharmaceutical carrier may be administered to a subject to
treat or prevent a gastrointestinal disorder including, but not
limited to, those provided above.
[0127] In still another embodiment, an agonist which modulates the
activity of HUTRAN may be administered to a subject to treat or
prevent a gastrointestinal disorder including, but not limited to,
those listed above.
[0128] In another embodiment, an antagonist of HUTRAN may be
administered to a subject to treat or prevent a cancer. Such a
cancer may include, but is not limited to, adenocarcinoma,
leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma,
and, in particular, cancers of the adrenal gland, bladder, bone,
bone marrow, brain, breast, cervix, gall bladder, ganglia,
gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary,
pancreas, parathyroid, penis, prostate, salivary glands, skin,
spleen, testis, thymus, thyroid, and uterus. In one aspect, an
antibody which specifically binds HUTRAN 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
HUTRAN.
[0129] In an additional embodiment, a vector expressing the
complement of the polynucleotide encoding HUTRAN may be
administered to a subject to treat or prevent a cancer including,
but not limited to, those described above.
[0130] In other embodiments, any of the proteins, antagonists,
antibodies, agonists, complementary sequences, or vectors of the
invention may be administered in combination with other appropriate
therapeutic agents. Selection of the appropriate agents for use in
combination therapy may be made by one of ordinary skill in the
art, according to conventional pharmaceutical principles. The
combination of therapeutic agents may act synergistically to effect
the treatment or prevention of the various disorders described
above. Using this approach, one may be able to achieve therapeutic
efficacy with lower dosages of each agent, thus reducing the
potential for adverse side effects.
[0131] An antagonist of HUTRAN may be produced using methods which
are generally known in the art. In particular, purified HUTRAN may
be used to produce antibodies or to screen libraries of
pharmaceutical agents to identify those which specifically bind
HUTRAN. Antibodies to HUTRAN may also be generated using methods
that are well known in the art. Such antibodies may include, but
are not limited to, polyclonal, monoclonal, chimeric, and single
chain antibodies, Fab fragments, and fragments produced by a Fab
expression library. Neutralizing antibodies (i.e., those which
inhibit dimer formation) are especially preferred for therapeutic
use.
[0132] For the production of polyclonal antibodies, various hosts
including goats, rabbits, rats, mice, humans, and others may be
immunized by injection with HUTRAN or with any fragment or
oligopeptide thereof which has immunogenic properties. Rats and
mice are preferred hosts for downstream applications involving
monoclonal antibody production. 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 aluminum hydroxide, and surface active substances such
as lysolecithin, pluronic polyols, polyanions, peptides, oil
emulsions, KLH, and dinitrophenol. Among adjuvants used in humans,
BCG (bacilli Calmette-Guerin) and Corynebacterium parvum are
especially preferable. (For review of methods for antibody
production and analysis, see, e.g., Harlow, E. and Lane, D. (1988)
Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory,
Cold Spring Harbor, N.Y.)
[0133] It is preferred that the oligopeptides, peptides, or
fragments used to induce antibodies to HUTRAN have an amino acid
sequence consisting of at least about 5 amino acids, and, more
preferably, of at least about 14 amino acids. It is also preferable
that these oligopeptides, peptides, or fragments are identical to a
portion of the amino acid sequence of the natural protein and
contain the entire amino acid sequence of a small, naturally
occurring molecule. Short stretches of HUTRAN amino acids may be
fused with those of another protein, such as KLH, and antibodies to
the chimeric molecule may be produced.
[0134] Monoclonal antibodies to HUTRAN 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. (See, e.g., 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. (1983) Proc. Natl.
Acad. Sci. 80:2026-2030; and Cole, S. P. et al. (1984) Mol. Cell
Biol. 62:109-120.)
[0135] In addition, techniques developed for the production of
"chimeric antibodies," such as the splicing of mouse antibody genes
to human antibody genes to obtain a molecule with appropriate
antigen specificity and biological activity, can be used. (See,
e.g., Morrison, S. L. et al. (1984) Proc. Natl. Acad. Sci.
81:6851-6855; Neuberger, M. S. et al. (1984) Nature 312:604-608;
and 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
HUTRAN-specific single chain antibodies. Antibodies with related
specificity, but of distinct idiotypic composition, may be
generated by chain shuffling from random combinatorial
immunoglobulin libraries. (See, e.g., Burton D. R. (1991) Proc.
Natl. Acad. Sci. 88:10134-10137.)
[0136] Antibodies may also be produced by inducing in vivo
production in the lymphocyte population or by screening
immunoglobulin libraries or panels of highly specific binding
reagents as disclosed in the literature. (See, e.g., Orlandi, R. et
al. (1989) Proc. Natl. Acad. Sci. 86: 3833-3837; and Winter, G. et
al. (1991) Nature 349:293-299.)
[0137] Antibody fragments which contain specific binding sites for
HUTRAN may also be generated. For example, such fragments include,
but are not limited to, F(ab')2 fragments produced by pepsin
digestion of the antibody molecule and Fab fragments generated by
reducing the disulfide bridges of the F(ab')2 fragments.
Alternatively, Fab expression libraries may be constructed to allow
rapid and easy identification of monoclonal Fab fragments with the
desired specificity. (See, e.g., Huse, W. D. et al. (1989) Science
246:1275-1281.)
[0138] Various immunoassays may be used for screening to identify
antibodies having the desired specificity and minimal
cross-reactivity. Numerous protocols for competitive binding or
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 HUTRAN and its specific antibody. A two-site,
monoclonal-based immunoassay utilizing monoclonal antibodies
reactive to two non-interfering HUTRAN epitopes is preferred, but a
competitive binding assay may also be employed. (Maddox,
supra.)
[0139] Various methods such as Scatchard analysis in conjunction
with radioimmunoassay techniques may be used to assess the affinity
of antibodies for HUTRAN. Affinity is expressed as an association
constant, K.sub.a, which is defined as the molar concentration of
HUTRAN-antibody complex divided by the molar concentrations of free
antigen and free antibody under equilibrium conditions. The K.sub.a
determined for a preparation of polyclonal antibodies, which are
heterogeneous in their affinities for multiple HUTRAN epitopes,
represents the average affinity, or avidity, of the antibodies for
HUTRAN. The K.sub.a determined for a preparation of monoclonal
antibodies, which are monospecific for a particular HUTRAN epitope,
represents a true measure of affinity. High-affinity antibody
preparations with K.sub.a ranging from about 10.sup.9 to 10.sup.12
L/mole are preferred for use in immunoassays in which the
HUTRAN-antibody complex must withstand rigorous manipulations.
Low-affinity antibody preparations with K.sub.a ranging from about
10.sup.6 to 10.sup.7 L/mole are preferred for use in
immunopurification and similar procedures which ultimately require
dissociation of HUTRAN, preferably in active form, from the
antibody. (Catty, D. (1988) Antibodies, Volume I: A Practical
Approach, IRL Press, Washington, D.C.; and Liddell, J. E. and
Cryer, A. (1991) A Practical Guide to Monoclonal Antibodies, John
Wiley & Sons, New York, N.Y.)
[0140] The titre and avidity of polyclonal antibody preparations
may be further evaluated to determine the quality and suitability
of such preparations for certain downstream applications. For
example, a polyclonal antibody preparation containing at least 1-2
mg specific antibody/ml, preferably 5-10 mg specific antibody/ml,
is preferred for use in procedures requiring precipitation of
HUTRAN-antibody complexes. Procedures for evaluating antibody
specificity, titer, and avidity, and guidelines for antibody
quality and usage in various applications, are generally available.
(See, e.g., Catty, supra, and Coligan et al. supra.)
[0141] In another embodiment of the invention, the polynucleotides
encoding HUTRAN, or any fragment or complement thereof, may be used
for therapeutic purposes. In one aspect, the complement of the
polynucleotide encoding HUTRAN 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 HUTRAN. Thus, complementary molecules
or fragments may be used to modulate HUTRAN activity, or to achieve
regulation of gene function. Such technology is now well known in
the art, and sense or antisense oligonucleotides or larger
fragments can be designed from various locations along the coding
or control regions of sequences encoding HUTRAN.
[0142] Expression vectors derived from retroviruses, adenoviruses,
or 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 vectors to
express nucleic acid sequences complementary to the polynucleotides
encoding HUTRAN. (See, e.g., Sambrook, supra; and Ausubel,
supra.)
[0143] Genes encoding HUTRAN can be turned off by transforming a
cell or tissue with expression vectors which express high levels of
a polynucleotide, or fragment thereof, encoding HUTRAN. 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 may last even longer if appropriate replication
elements are part of the vector system.
[0144] As mentioned above, modifications of gene expression can be
obtained by designing complementary sequences or antisense
molecules (DNA, RNA, or PNA) to the control, 5', or regulatory
regions of the gene encoding HUTRAN. Oligonucleotides derived from
the transcription initiation site, e.g., between about 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 causes
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. (See, e.g., 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., pp.
163-177.) A complementary sequence or antisense molecule may also
be designed to block translation of mRNA by preventing the
transcript from binding to ribosomes.
[0145] Ribozymes, enzymatic RNA molecules, may also be used to
catalyze the specific cleavage of RNA. The mechanism of ribozyme
action involves sequence-specific hybridization of the ribozyme
molecule to complementary target RNA, followed by endonucleolytic
cleavage. For example, engineered hammerhead motif ribozyme
molecules may specifically and efficiently catalyze endonucleolytic
cleavage of sequences encoding HUTRAN.
[0146] Specific ribozyme cleavage sites within any potential RNA
target are initially identified by scanning the target molecule for
ribozyme cleavage sites, including 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 hybridization with
complementary oligonucleotides using ribonuclease protection
assays.
[0147] Complementary ribonucleic acid 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 synthesizing 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 HUTRAN. Such DNA sequences may be incorporated
into a wide variety of vectors with suitable RNA polymerase
promoters such as T7 or SP6. Alternatively, these cDNA constructs
that synthesize complementary RNA, constitutively or inducibly, can
be introduced into cell lines, cells, or tissues.
[0148] 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
nontraditional 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 recognized by endogenous endonucleases.
[0149] 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,
by liposome injections, or by polycationic amino polymers may be
achieved using methods which are well known in the art. (See, e.g.,
Goldman, C. K. et al. (1997) Nature Biotechnology 15:462-466.)
[0150] Any of the therapeutic methods described above may be
applied to any subject in need of such therapy, including, for
example, mammals such as dogs, cats, cows, horses, rabbits,
monkeys, and most preferably, humans.
[0151] An additional embodiment of the invention relates to the
administration of a pharmaceutical or sterile composition, in
conjunction with a pharmaceutically acceptable carrier, for any of
the therapeutic effects discussed above. Such pharmaceutical
compositions may consist of HUTRAN, antibodies to HUTRAN, and
mimetics, agonists, antagonists, or inhibitors of HUTRAN. The
compositions may be administered alone or in combination with at
least one other agent, such as a stabilizing 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.
[0152] The pharmaceutical compositions utilized 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.
[0153] 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.).
[0154] 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.
[0155] Pharmaceutical preparations for oral use can be obtained
through combining active compounds with solid excipient and
processing the resultant mixture of granules (optionally, after
grinding) to obtain tablets or dragee cores. Suitable auxiliaries
can be added, if desired. Suitable excipients include carbohydrate
or protein fillers, such as sugars, including lactose, sucrose,
mannitol, and sorbitol; starch from corn, wheat, rice, potato, or
other plants; cellulose, such as methyl cellulose,
hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose;
gums, including arabic and tragacanth; and proteins, such as
gelatin and collagen. If desired, disintegrating or solubilizing
agents may be added, such as the cross-linked polyvinyl
pyrrolidone, agar, and alginic acid or a salt thereof, such as
sodium alginate.
[0156] Dragee cores may be used in conjunction with suitable
coatings, such as concentrated sugar solutions, which may also
contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel,
polyethylene glycol, and/or titanium dioxide, lacquer solutions,
and suitable organic solvents or solvent mixtures. Dyestuffs or
pigments may be added to the tablets or dragee coatings for product
identification or to characterize the quantity of active compound,
i.e., dosage.
[0157] Pharmaceutical preparations which can be used orally include
push-fit capsules made of gelatin, as well as soft, sealed capsules
made of gelatin and a coating, such as glycerol or sorbitol.
Push-fit capsules can contain active ingredients mixed with fillers
or binders, such as lactose or starches, lubricants, such as talc
or magnesium stearate, and, optionally, stabilizers. In soft
capsules, the active compounds may be dissolved or suspended in
suitable liquids, such as fatty oils, liquid, or liquid
polyethylene glycol with or without stabilizers.
[0158] Pharmaceutical formulations suitable for parenteral
administration may be formulated in aqueous solutions, preferably
in physiologically compatible buffers such as Hanks' solution,
Ringer's solution, or physiologically buffered saline. Aqueous
injection suspensions may contain substances which increase the
viscosity of the suspension, such as sodium carboxymethyl
cellulose, sorbitol, or dextran. Additionally, suspensions of the
active compounds may be prepared as appropriate oily injection
suspensions. Suitable lipophilic solvents or vehicles include fatty
oils, such as sesame oil, or synthetic fatty acid esters, such as
ethyl oleate, triglycerides, or liposomes. Non-lipid polycationic
amino polymers may also be used for delivery. Optionally, the
suspension may also contain suitable stabilizers or agents to
increase the solubility of the compounds and allow for the
preparation of highly concentrated solutions.
[0159] For topical or nasal administration, penetrants appropriate
to the particular barrier to be permeated are used in the
formulation. Such penetrants are generally known in the art.
[0160] 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 lyophilizing processes.
[0161] The pharmaceutical composition may be provided as a salt and
can be formed with many acids, including but not limited to,
hydrochloric, sulfuric, acetic, lactic, tartaric, malic, and
succinic acid. Salts tend to be more soluble in aqueous or other
protonic solvents than are the corresponding free base forms. In
other cases, the preferred preparation may be a lyophilized powder
which may contain any or all of the following: 1 mM to 50 mM
histidine, 0.1% to 2% sucrose, and 2% to 7% mannitol, at a pH range
of 4.5 to 5.5, that is combined with buffer prior to use.
[0162] After pharmaceutical compositions have been prepared, they
can be placed in an appropriate container and labeled for treatment
of an indicated condition. For administration of HUTRAN, such
labeling would include amount, frequency, and method of
administration.
[0163] 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.
[0164] For any compound, the therapeutically effective dose can be
estimated initially either in cell culture assays, e.g., of
neoplastic cells or in animal models such as mice, rats, rabbits,
dogs, or pigs. An 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.
[0165] A therapeutically effective dose refers to that amount of
active ingredient, for example HUTRAN or fragments thereof,
antibodies of HUTRAN, and agonists, antagonists or inhibitors of
HUTRAN, which ameliorates the symptoms or condition. Therapeutic
efficacy and toxicity may be determined by standard pharmaceutical
procedures in cell cultures or with experimental animals, such as
by calculating the ED.sub.50 (the dose therapeutically effective in
50% of the population) or LD.sub.50 (the dose lethal to 50% of the
population) statistics. The dose ratio of therapeutic to toxic
effects is the therapeutic index, and it can be expressed as the
ED.sub.50/LD.sub.50 ratio. Pharmaceutical compositions which
exhibit large therapeutic indices are preferred. The data obtained
from cell culture assays and animal studies are used to formulate a
range of dosage for human use. The dosage contained in such
compositions is preferably within a range of circulating
concentrations that includes the ED.sub.50 with little or no
toxicity. The dosage varies within this range depending upon the
dosage form employed, the sensitivity of the patient, and the route
of administration.
[0166] The exact dosage will be determined by the practitioner, in
light of factors related to the subject requiring 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,
the general health of the subject, the age, weight, and gender of
the subject, time and frequency of administration, drug
combination(s), reaction sensitivities, and response to therapy.
Long-acting pharmaceutical compositions may be administered every 3
to 4 days, every week, or biweekly depending on the half-life and
clearance rate of the particular formulation.
[0167] Normal dosage amounts may vary from about 0.1 .mu.g to
100,000 .mu.g, up to a total dose of about 1 gram, 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
will 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.
[0168] Diagnostics
[0169] In another embodiment, antibodies which specifically bind
HUTRAN may be used for the diagnosis of disorders characterized by
expression of HUTRAN, or in assays to monitor patients being
treated with HUTRAN or agonists, antagonists, or inhibitors of
HUTRAN. Antibodies useful for diagnostic purposes may be prepared
in the same manner as described above for therapeutics. Diagnostic
assays for HUTRAN include methods which utilize the antibody and a
label to detect HUTRAN in human body fluids or in extracts of cells
or tissues. The antibodies may be used with or without
modification, and may be labeled by covalent or non-covalent
attachment of a reporter molecule. A wide variety of reporter
molecules, several of which are described above, are known in the
art and may be used.
[0170] A variety of protocols for measuring HUTRAN, including
ELISAs, RIAs, and FACS, are known in the art and provide a basis
for diagnosing altered or abnormal levels of HUTRAN expression.
Normal or standard values for HUTRAN expression are established by
combining body fluids or cell extracts taken from normal mammalian
subjects, preferably human, with antibody to HUTRAN under
conditions suitable for complex formation. The amount of standard
complex formation may be quantitated by various methods, preferably
by photometric means. Quantities of HUTRAN expressed in subject
samples from biopsied tissues are compared with the standard
values. Deviation between standard and subject values establishes
the parameters for diagnosing disease.
[0171] In another embodiment of the invention, the polynucleotides
encoding HUTRAN may be used for diagnostic purposes. The
polynucleotides which may be used include oligonucleotide
sequences, complementary RNA and DNA molecules, and PNAs. The
polynucleotides may be used to detect and quantitate gene
expression in biopsied tissues in which expression of HUTRAN may be
correlated with disease. The diagnostic assay may be used to
determine absence, presence, and excess expression of HUTRAN, and
to monitor regulation of HUTRAN levels during therapeutic
intervention.
[0172] In one aspect, hybridization with PCR probes which are
capable of detecting polynucleotide sequences, including genomic
sequences, encoding HUTRAN or closely related molecules may be used
to identify nucleic acid sequences which encode HUTRAN. The
specificity of the probe, whether it is made from a highly specific
region, e.g., the 5' regulatory region, or from a less specific
region, e.g., a conserved motif, and the stringency of the
hybridization or amplification (maximal, high, intermediate, or
low), will determine whether the probe identifies only naturally
occurring sequences encoding HUTRAN, allelic variants, or related
sequences.
[0173] Probes may also be used for the detection of related
sequences, and should preferably have at least 50% sequence
identity to any of the HUTRAN encoding sequences. The hybridization
probes of the subject invention may be DNA or RNA and may be
derived from the sequence of the sequences of SEQ ID NO:4, SEQ ID
NO:5, or SEQ ID NO:6 or from genomic sequences including promoters,
enhancers, and introns of the HUTRAN gene.
[0174] Means for producing specific hybridization probes for DNAs
encoding HUTRAN include the cloning of polynucleotide sequences
encoding HUTRAN or HUTRAN derivatives into vectors for the
production of mRNA probes. Such vectors are known in the art, are
commercially available, and may be used to synthesize RNA probes in
vitro by means of the addition of the appropriate RNA polymerases
and the appropriate labeled nucleotides. Hybridization probes may
be labeled by a variety of reporter groups, for example, by
radionuclides such as .sup.32P or .sup.35S, or by enzymatic labels,
such as alkaline phosphatase coupled to the probe via avidin/biotin
coupling systems, and the like.
[0175] Polynucleotide sequences encoding HUTRAN may be used for the
diagnosis of a disorder associated with expression of HUTRAN.
Examples of such a disorder include, but are not limited to,
autoimmune/inflammatory disorders such as acquired immunodeficiency
syndrome (AIDS), Addison's disease, adult respiratory distress
syndrome, allergies, ankylosing spondylitis, amyloidosis, anemia,
asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune
thyroiditis, bronchitis, cholecystitis, contact dermatitis, Crohn's
disease, atopic dermatitis, dermatomyositis, diabetes mellitus,
emphysema, episodic lymphopenia with lymphocytotoxins,
erythroblastosis fetalis, erythema nodosum, atrophic gastritis,
glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease,
Hashimoto's thyroiditis, hypereosinophilia, irritable bowel
syndrome, multiple sclerosis, myasthenia gravis, myocardial or
pericardial inflammation, osteoarthritis, osteoporosis,
pancreatitis, polymyositis, psoriasis, Reiter's syndrome,
rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic
anaphylaxis, systemic lupus erythematosus, systemic sclerosis,
thrombocytopenic purpura, ulcerative colitis, uveitis, Werner
syndrome, complications of cancer, hemodialysis, and extracorporeal
circulation, viral, bacterial, fungal, parasitic, protozoal, and
helminthic infections, and trauma; neurological disorders such as
epilepsy, ischemic cerebrovascular disease, stroke, cerebral
neoplasms, Alzheimer's disease, Pick's disease, Huntington's
disease, dementia, Parkinson's disease and other extrapyramidal
disorders, amyotrophic lateral sclerosis and other motor neuron
disorders, progressive neural muscular atrophy, retinitis
pigmentosa, hereditary ataxias, multiple sclerosis and other
demyelinating diseases, bacterial and viral meningitis, brain
abscess, subdural empyema, epidural abscess, suppurative
intracranial thrombophlebitis, myelitis and radiculitis, viral
central nervous system disease; prion diseases including kuru,
Creutzfeldt-Jakob disease, and Gerstmann-Straussler-Scheinker
syndrome; fatal familial insomnia, nutritional and metabolic
diseases of the nervous system, neurofibromatosis, tuberous
sclerosis, cerebelloretinal hemangioblastomatosis,
encephalotrigeminal syndrome, mental retardation and other
developmental disorders of the central nervous system, cerebral
palsy, neuroskeletal disorders, autonomic nervous system disorders,
cranial nerve disorders, spinal cord diseases, muscular dystrophy
and other neuromuscular disorders, peripheral nervous system
disorders, dermatomyositis and polymyositis; inherited, metabolic,
endocrine, and toxic myopathies; myasthenia gravis, periodic
paralysis; mental disorders including mood, anxiety, and
schizophrenic disorders; akathesia, amnesia, catatonia, diabetic
neuropathy, tardive dyskinesia, dystonias, paranoid psychoses,
postherpetic neuralgia, and Tourette's disorder; reproductive
disorders such as disorders of prolactin production; infertility,
including tubal disease, ovulatory defects, and endometriosis;
disruptions of the estrous cycle, disruptions of the menstrual
cycle, polycystic ovary syndrome, ovarian hyperstimulation
syndrome, endometrial and ovarian tumors, uterine fibroids,
autoimmune disorders, ectopic pregnancies, and teratogenesis;
cancer of the breast, fibrocystic breast disease, and galactorrhea;
disruptions of spermatogenesis, abnormal sperm physiology, cancer
of the testis, cancer of the prostate, benign prostatic
hyperplasia, prostatitis, Peyronie's disease, carcinoma of the male
breast, and gynecomastia; gastrointestinal disorders such as;
dysphagia, peptic esophagitis, esophageal spasm, esophageal
stricture, esophageal carcinoma, dyspepsia, indigestion, gastritis,
gastric carcinoma, anorexia, nausea, emesis, gastroparesis, antral
or pyloric edema, abdominal angina, pyrosis, gastroenteritis,
intestinal obstruction, infections of the intestinal tract, peptic
ulcer, cholelithiasis, cholecystitis, cholestasis, pancreatitis,
pancreatic carcinoma, biliary tract disease, hepatitis,
hyperbilirubinemia, cirrhosis, passive congestion of the liver,
hepatoma, infectious colitis, ulcerative colitis, ulcerative
proctitis, Crohn's disease, Whipple's disease, Mallory-Weiss
syndrome, colonic carcinoma, colonic obstruction, irritable bowel
syndrome, short bowel syndrome, diarrhea, constipation,
gastrointestinal hemorrhage, and acquired immunodeficiency syndrome
(AIDS) enteropathy; and cancers such as adenocarcinoma, leukemia,
lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in
particular, cancers of the adrenal gland, bladder, bone, bone
marrow, brain, breast, cervix, gall bladder, ganglia,
gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary,
pancreas, parathyroid, penis, prostate, salivary glands, skin,
spleen, testis, thymus, thyroid, and uterus. The polynucleotide
sequences encoding HUTRAN may be used in Southern or Northern
analysis, dot blot, or other membrane-based technologies; in PCR
technologies; in dipstick, pin, and ELISA assays; and in
microarrays utilizing fluids or tissues from patients to detect
altered HUTRAN expression. Such qualitative or quantitative methods
are well known in the art.
[0176] In a particular aspect, the nucleotide sequences encoding
HUTRAN may be useful in assays that detect the presence of
associated disorders, particularly those mentioned above. The
nucleotide sequences encoding HUTRAN may be labeled by standard
methods and added to a fluid or tissue sample from a patient under
conditions suitable for the formation of hybridization complexes.
After a suitable incubation period, the sample is washed and the
signal is quantitated and compared with a standard value. If the
amount of signal in the patient sample is significantly altered in
comparison to a control sample then the presence of altered levels
of nucleotide sequences encoding HUTRAN in the sample indicates the
presence of the associated disorder. Such assays may also be used
to evaluate the efficacy of a particular therapeutic treatment
regimen in animal studies, in clinical trials, or to monitor the
treatment of an individual patient.
[0177] In order to provide a basis for the diagnosis of a disorder
associated with expression of HUTRAN, 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,
encoding HUTRAN, under conditions suitable for hybridization or
amplification. Standard hybridization may be quantified by
comparing the values obtained from normal subjects with values from
an experiment in which a known amount of a substantially purified
polynucleotide is used. Standard values obtained in this manner may
be compared with values obtained from samples from patients who are
symptomatic for a disorder. Deviation from standard values is used
to establish the presence of a disorder.
[0178] Once the presence of a disorder is established and a
treatment protocol is initiated, hybridization assays may be
repeated on a regular basis to determine if the level of expression
in the patient begins to approximate that which is observed in the
normal subject. The results obtained from successive assays may be
used to show the efficacy of treatment over a period ranging from
several days to months.
[0179] With respect to cancer, the 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 cancer.
[0180] Additional diagnostic uses for oligonucleotides designed
from the sequences encoding HUTRAN may involve the use of PCR.
These oligomers may be chemically synthesized, generated
enzymatically, or produced in vitro. Oligomers will preferably
contain a fragment of a polynucleotide encoding HUTRAN, or a
fragment of a polynucleotide complementary to the polynucleotide
encoding HUTRAN, and will be employed under optimized conditions
for identification of a specific gene or condition. Oligomers may
also be employed under less stringent conditions for detection or
quantitation of closely related DNA or RNA sequences.
[0181] Methods which may also be used to quantitate the expression
of HUTRAN include radiolabeling or biotinylating nucleotides,
coamplification of a control nucleic acid, and interpolating
results from standard curves. (See, e.g., Melby, P. C. et al.
(1993) J. Immunol. Methods 159:235-244; and Duplaa, C. et al.
(1993) Anal. Biochem. 229-236.) The speed of quantitation 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 quantitation.
[0182] In further embodiments, oligonucleotides or longer fragments
derived from any of the polynucleotide sequences described herein
may be used as targets in a microarray. The microarray can be used
to monitor the expression level of large numbers of genes
simultaneously and to identify genetic variants, mutations, and
polymorphisms. This information may be used to determine gene
function, to understand the genetic basis of a disorder, to
diagnose a disorder, and to develop and monitor the activities of
therapeutic agents.
[0183] Microarrays may be prepared, used, and analyzed using
methods known in the art. (See, e.g., Brennan, T. M. et al. (1995)
U.S. Pat. No. 5,474,796; Schena, M. et al. (1996) Proc. Natl. Acad.
Sci. 93:10614-10619; Baldeschweiler et al. (1995) PCT application
WO95/251116; Shalon, D. et al. (1995) PCT application WO95/35505;
Heller, R. A. et al. (1997) Proc. Natl. Acad. Sci. 94:2150-2155;
and Heller, M. J. et al. (1997) U.S. Pat. No. 5,605,662.)
[0184] In another embodiment of the invention, nucleic acid
sequences encoding HUTRAN may be used to generate hybridization
probes useful in mapping the naturally occurring genomic sequence.
The sequences may be mapped to a particular chromosome, to a
specific region of a chromosome, or to artificial chromosome
constructions, e.g., human artificial chromosomes (HACs), yeast
artificial chromosomes (YACs), bacterial artificial chromosomes
(BACs), bacterial P1 constructions, or single chromosome cDNA
libraries. (See, e.g., Price, C. M. (1993) Blood Rev. 7:127-134;
and Trask, B. J. (1991) Trends Genet. 7:149-154.)
[0185] Fluorescent in situ hybridization (FISH) may be correlated
with other physical chromosome mapping techniques and genetic map
data. (See, e.g., Heinz-Ulrich, et al. (1995) in Meyers, R. A.
(ed.) Molecular Biology and Biotechnology, VCH Publishers New York,
N.Y., pp. 965-968.) Examples of genetic map data can be found in
various scientific journals or at the Online Mendelian Inheritance
in Man (OMIM) site. Correlation between the location of the gene
encoding HUTRAN on a physical chromosomal map and a specific
disorder, or a predisposition to a specific disorder, may help
define the region of DNA associated with that disorder. The
nucleotide sequences of the invention may be used to detect
differences in gene sequences among normal, carrier, and affected
individuals.
[0186] 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 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, e.g., ataxia-telangiectasia to 11q22-23, any
sequences mapping to that area may represent associated or
regulatory genes for further investigation. (See, e.g., Gatti, R.
A. et al. (1988) Nature 336:577-580.) The nucleotide sequence 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.
[0187] In another embodiment of the invention, HUTRAN, its
catalytic or immunogenic fragments, or oligopeptides thereof can be
used for screening libraries of compounds in any of a variety of
drug screening techniques. The fragment 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 HUTRAN and the agent being tested may be
measured.
[0188] Another technique for drug screening provides for high
throughput screening of compounds having suitable binding affinity
to the protein of interest. (See, e.g., Geysen, et al. (1984) PCT
application WO84/03564.) In this method, large numbers of different
small test compounds are synthesized on a solid substrate, such as
plastic pins or some other surface. The test compounds are reacted
with HUTRAN, or fragments thereof, and washed. Bound HUTRAN is then
detected by methods well known in the art. Purified HUTRAN 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.
[0189] In another embodiment, one may use competitive drug
screening assays in which neutralizing antibodies capable of
binding HUTRAN specifically compete with a test compound for
binding HUTRAN. In this manner, antibodies can be used to detect
the presence of any peptide which shares one or more antigenic
determinants with HUTRAN.
[0190] In additional embodiments, the nucleotide sequences which
encode HUTRAN may be used in any molecular biology techniques that
have yet to be developed, provided the new techniques rely on
properties of nucleotide sequences that are currently known,
including, but not limited to, such properties as the triplet
genetic code and specific base pair interactions.
[0191] The examples below are provided to illustrate the subject
invention and are not included for the purpose of limiting the
invention.
EXAMPLES
[0192] 1. Construction of cDNA Libraries
[0193] RNA was isolated from tissues described in Table 4. Some
tissues were homogenized and lysed in guanidinium isothiocyanate,
while others were homogenized and lysed in phenol or in a suitable
mixture of denaturants, such as TRIZOL (Life Technologies, Inc.,
Gaithersburg, Md.), a monophasic solution of phenol and guanidine
isothiocyanate. The resulting lysates were centrifuged over CsCl
cushions or extracted with chloroform. RNA was precipitated from
the lysates with either isopropanol or sodium acetate and ethanol,
or by other routine methods.
[0194] Phenol extraction and precipitation of RNA were repeated as
necessary to increase RNA purity. In some cases, RNA was treated
with DNase. For most libraries, poly(A+) RNA was isolated using
oligo d(T)-coupled paramagnetic particles (Promega Corp., Madison,
Wis.), OLIGOTEX latex particles (QIAGEN Inc., Valencia, Calif.), or
an OLIGOTEX mRNA purification kit (QIAGEN Inc., Valencia, Calif.).
Alternatively, RNA was isolated directly from tissue lysates using
other RNA isolation kits, e.g., the POLY(A)PURE mRNA purification
kit (Ambion, Austin, Tex.).
[0195] cDNA was synthesized and cDNA libraries were constructed
with the SUPERSCRIPT plasmid system (Life Technologies, Inc.,
Gaithersburg, Md.), using the recommended procedures or similar
methods known in the art. (See, e.g., Ausubel, supra, 1997, units
5.1-6.6) Reverse transcription was initiated using oligo d(T) or
random primers. Synthetic oligonucleotide adapters were ligated to
double stranded cDNA, and the cDNA was digested with the
appropriate restriction enzyme or enzymes. For most libraries, the
cDNA was size-selected (300-1000 bp) using SEPHACRYL S1000,
SEPHAROSE CL2B, or SEPHAROSE CL4B column chromatography (Amersham
Pharmacia Biotech, Piscataway, N.J.) or preparative agarose gel
electrophoresis. cDNAs were ligated into compatible restriction
enzyme sites of the polylinker of pINCY (Incyte Pharmaceuticals,
Inc., Palo Alto, Calif.). Recombinant plasmids were transformed
into competent E. coli cells, e.g., the XL1-Blue, XL1-BlueMRF, or
SOLR strains (Stratagene, Inc., La Jolla, Calif.), or DH5.alpha.,
DH10B, or ElectroMAX DH10B (Life Technologies, Inc., Gaithersburg,
Md.).
[0196] II. Isolation of cDNA Clones
[0197] Plasmids were recovered from host cells by cell lysis.
Plasmids were purified using at least one of the following: a Magic
or WIZARD Minipreps DNA purification system (Promega Corp.,
Madison, Wis.); an AGTC Miniprep purification kit (Edge Biosystems,
Gaithersburg, Md.); the QIAWELL 8 Plasmid, QIAWELL 8 Plus Plasmid,
or the QIAWELL 8 Ultra Plasmid purification systems (QIAGEN Inc.,
Valencia, Calif.); or the R.E.A.L. PREP 96 plasmid kit (QIAGEN
Inc., Valencia, Calif.). Following precipitation, plasmids were
resuspended in 0.1 ml of distilled water and stored, with or
without lyophilization, at 4.degree. C.
[0198] Alternatively, plasmid DNA was amplified from host cell
lysates using direct link PCR in a high-throughput format. (Rao, V.
B. (1994) Anal. Biochem. 216:1-14.) Host cell lysis and thermal
cycling steps were carried out in a single reaction mixture.
Samples were processed and stored in 384-well plates, and the
concentration of amplified plasmid DNA was quantified
fluorometrically using PICOGREEN dye (Molecular Probes, Inc.,
Eugene, Oreg.) and a Fluoroskan II fluorescence scanner (Labsystems
Oy, Helsinki, Finland).
[0199] III. Sequencing and Analysis
[0200] The cDNAs were prepared for sequencing using either an ABI
CATALYST 800 thermal cycler (Perkin Elmer), or a MICROLAB 2200
liquid transfer system (Hamilton, Reno, Nev.) in combination with
PTC200 thermal cyclers (MJ Research, Watertown Mass.). The cDNAs
were sequenced on the ABI 373 or 377 DNA sequencing systems (Perkin
Elmer) by the method of Sanger F. and A. R. Coulson (1975; J. Mol.
Biol. 94:441-448) using standard ABI protocols, base calling
software, and kits. Alternatively, cDNAs were sequenced using
solutions and dyes from Amersham Pharmacia Biotech. Reading frame
was determined using standard methods (Ausubel, supra).
[0201] The cDNA sequences presented in Table I and the full length
nucleotide and amino acid sequences disclosed in the Sequence
Listing were queried against databases such as GenBank primate
(pri), rodent (rod), mammalian (mamp), vertebrate (vrtp), and
eukaryote (eukp) databases, SwissProt, BLOCKS, and other databases
which contain previously identified and annotated motifs and
sequences. Algorithms such as Smith Waterman which deal with
primary sequence patterns and secondary structure gap penalties
(Smith, T. et al. (1992) Protein Engineering 5:35-51) and programs
and algorithms such as BLAST (Basic Local Alignment Search Tool;
Altschul, S. F. (1993) J. Mol. Evol 36:290-300; and Altschul et al.
(1990) J. Mol. Biol. 215:403-410), and HMM (Hidden Markov Models;
Eddy, S. R. (1996) Cur. Opin. Str. Biol. 6:361-365 and Sonnhammer,
E. L. L. et al. (1997) Proteins 28:405-420) were used to assemble
and analyze nucleotide and amino acid sequences. The databases,
programs, algorithms, methods and tools are available, well known
in the art, and described in Ausubel (supra, unit 7.7), in Meyers,
R. A. (1995; Molecular Biology and Biotechnology, Wiley VCH, Inc,
New York N.Y., p 856-853), in documentation provided with software
(Genetics Computer Group (GCG), Madison Wis.), and on the world
wide web (www). Two comprehensive websites which list, describe,
and/or link many of the databases and tools are: 1) the www
resource in practical sequence analysis (http://genome.wustl.edu/),
and 2) the bibliography of computational gene recognition
(http://linkage.rockefeller.edu/wli/gene/programs.html). For
example, the first website links PFAM as a database
(http://genome.wustl.edu/Pfam/) and as an HMM search tool
(http://genome.wustl.edu/eddy/cgi-bin/hmm_page.cgi).
[0202] Table 5 summarizes the databases and tools used herein.
[0203] IV. Northern Analysis
[0204] Northern analysis is a laboratory technique used to detect
the presence of a transcript of a gene and involves the
hybridization of a labeled nucleotide sequence to a membrane on
which RNAs from a particular cell type or tissue have been bound.
(See, e.g., Sambrook, supra, ch. 7; and Ausubel, supra, ch. 4 and
16.)
[0205] Analogous computer techniques applying BLAST are used to
search for identical or related molecules in nucleotide databases
such as GenBank or LIFESEQ database (Incyte Pharmaceuticals). This
analysis is much faster than multiple membrane-based
hybridizations. In addition, the sensitivity of the computer search
can be modified to determine whether any particular match is
categorized as exact or similar.
[0206] The basis of the search is the product score, which is
defined as: 1 % sequence identity .times. % maximum BLAST score
100
[0207] The product score takes into account both the degree of
similarity between two sequences and the length of the sequence
match. For example, with a product score of 40, the match will be
exact within a 1% to 2% error, and, with a product score of 70, the
match will be exact. Similar molecules are usually identified by
selecting those which show product scores between 15 and 40,
although lower scores may identify related molecules.
[0208] The results of Northern analysis are reported as a list of
libraries in which the transcript encoding HUTRAN occurs. Abundance
and percent abundance are also reported. Abundance directly
reflects the number of times a particular transcript is represented
in a cDNA library, and percent abundance is abundance divided by
the total number of sequences examined in the cDNA library.
[0209] V. Extension of HUTRAN Encoding Polynucleotides
[0210] The nucleic acid sequences of Incyte Clones 1815528,
2150892, and 2525071 were used to design oligonucleotide primers
for extending partial nucleotide sequences to full length. For each
nucleic acid sequence, one primer was synthesized to initiate
extension of an antisense polynucleotide, and the other was
synthesized to initiate extension of a sense polynucleotide.
Primers were used to facilitate the extension of the known sequence
"outward" generating amplicons containing new unknown nucleotide
sequence for the region of interest. The initial primers were
designed from the cDNA using OLIGO 4.06 software (National
Biosciences, Plymouth, Minn.), or another appropriate program, to
be about 22 to 30 nucleotides in length, to have a GC content of
about 50% or more, and to anneal to the target sequence at
temperatures of about 68.degree. C. to about 72.degree. C. Any
stretch of nucleotides which would result in hairpin structures and
primer-primer dimerizations was avoided.
[0211] Selected human cDNA libraries (Life Technologies, Inc.,
Gaithersburg, Md.) were used to extend the sequence. If more than
one extension is necessary or desired, additional sets of primers
are designed to further extend the known region.
[0212] High fidelity amplification was obtained by following the
instructions for the XL-PCR kit (Perkin Elmer) and thoroughly
mixing the enzyme and reaction mix. PCR was performed using the
PTC200 thermal cycler (M.J. Research, Watertown, Mass.), beginning
with 40 pmol of each primer and the recommended concentrations of
all other components of the kit, with the following parameters:
1 Step 1 94.degree. C. for 1 min (initial denaturation) Step 2
65.degree. C. for 1 min Step 3 68.degree. C. for 6 min Step 4
94.degree. C. for 15 sec Step 5 65.degree. C. for 1 min Step 6
68.degree. C. for 7 min Step 7 Repeat steps 4 through 6 for an
additional 15 cycles Step 8 94.degree. C. for 15 sec Step 9
65.degree. C. for 1 min Step 10 68.degree. C. for 7:15 min Step 11
Repeat steps 8 through 10 for an additional 12 cycles Step 12
72.degree. C. for 8 min Step 13 4.degree. C. (and holding)
[0213] A 5 .mu.l to 10 .mu.l aliquot of the reaction mixture was
analyzed by electrophoresis on a low concentration (about 0.6% to
0.8%) agarose mini-gel to determine which reactions were successful
in extending the sequence. Bands thought to contain the largest
products were excised from the gel, purified using QIAQUICK (QIAGEN
Inc.), and trimmed of overhangs using Klenow enzyme to facilitate
religation and cloning.
[0214] After ethanol precipitation, the products were redissolved
in 13 .mu.l of ligation buffer, 1 .mu.l T4-DNA ligase (15 units)
and 1 .mu.l T4 polynucleotide kinase were added, and the mixture
was incubated at room temperature for 2 to 3 hours, or overnight at
16.degree. C. Competent E. coli cells (in 40 .mu.l of appropriate
media) were transformed with 3 .mu.l of ligation mixture and
cultured in 80 .mu.l of SOC medium. (See, e.g., Sambrook, supra,
Appendix A, p. 2.) After incubation for one hour at 37.degree. C.,
the E. coli mixture was plated on Luria Bertani (LB) agar (See,
e.g., Sambrook, supra, Appendix A, p. 1) containing carbenicillin
(2.times.carb). The following day, several colonies were randomly
picked from each plate and cultured in 150 .mu.l of liquid
LB/2.times.carb medium placed in an individual well of an
appropriate commercially-available sterile 96-well microtiter
plate. The following day, 5 .mu.l of each overnight culture was
transferred into a non-sterile 96-well plate and, after dilution
1:10 with water, 5 .mu.l from each sample was transferred into a
PCR array.
[0215] For PCR amplification, 18 .mu.l of concentrated PCR reaction
mix (3.3.times.) containing 4 units of rTth DNA polymerase, a
vector primer, and one or both of the gene specific primers used
for the extension reaction were added to each well. Amplification
was performed using the following conditions:
2 Step 1 94.degree. C. for 60 sec Step 2 94.degree. C. for 20 sec
Step 3 55.degree. C. for 30 sec Step 4 72.degree. C. for 90 sec
Step 5 Repeat steps 2 through 4 for an additional 29 cycles Step 6
72.degree. C. for 180 sec Step 7 4.degree. C. (and holding)
[0216] Aliquots of the PCR reactions were run on agarose gels
together with molecular weight markers. The sizes of the PCR
products were compared to the original partial cDNAs, and
appropriate clones were selected, ligated into plasmid, and
sequenced.
[0217] In like manner, the nucleotide sequences of SEQ ID NO:4-6
are used to obtain 5' regulatory sequences using the procedure
above, oligonucleotides designed for 5' extension, and an
appropriate genomic library.
[0218] VI. Labeling and Use of Individual Hybridization Probes
[0219] Hybridization probes derived from SEQ ID NO:4-6 are employed
to screen cDNAs, genomic DNAs, or mRNAs. Although the labeling of
oligonucleotides, consisting of about 20 base pairs, is
specifically described, essentially the same procedure is used with
larger nucleotide fragments. Oligonucleotides are designed using
state-of-the-art software such as OLIGO 4.06 software (National
Biosciences) and labeled by combining 50 pmol of each oligomer, 250
.mu.Ci of [.gamma.-.sup.32P] adenosine triphosphate (Amersham,
Chicago, Ill.), and T4 polynucleotide kinase (DUPONT NEN, Boston,
Mass.). The labeled oligonucleotides are substantially purified
using a SEPHADEX G-25 superfine size exclusion dextran bead column
(Pharmacia & Upjohn, Kalamazoo, Mich.). An aliquot containing
10.sup.7 counts per minute of the labeled probe is used in a
typical membrane-based hybridization analysis of human genomic DNA
digested with one of the following endonucleases: Ase I, Bgl II,
Eco RI, Pst I, Xbal, or Pvu II (DuPont NEN, Boston, Mass.).
[0220] The DNA from each digest is fractionated on a 0.7% agarose
gel and transferred to nylon membranes (Nytran Plus, Schleicher
& Schuell, Durham, N.H.). Hybridization is carried out for 16
hours at 40.degree. C. To remove nonspecific signals, blots are
sequentially washed at room temperature under increasingly
stringent conditions up to 0.1.times.saline sodium citrate and 0.5%
sodium dodecyl sulfate. After XOMAT AR film (Kodak, Rochester,
N.Y.) is exposed to the blots, hybridization patterns are compared
visually.
[0221] VII. Microarrays
[0222] A chemical coupling procedure and an ink jet device can be
used to synthesize array elements on the surface of a substrate.
(See, e.g., Baldeschweiler, supra.) An array analogous to a dot or
slot blot may also be used to arrange and link elements to the
surface of a substrate using thermal, UV, chemical, or mechanical
bonding procedures. A typical array may be produced by hand or
using available methods and machines and contain any appropriate
number of elements. After hybridization, nonhybridized probes are
removed and a scanner used to determine the levels and patterns of
fluorescence. The degree of complementarity and the relative
abundance of each probe which hybridizes to an element on the
microarray may be assessed through analysis of the scanned
images.
[0223] Full-length cDNAs, Expressed Sequence Tags (ESTs), or
fragments thereof may comprise the elements of the microarray.
Fragments suitable for hybridization can be selected using software
well known in the art such as LASERGENE. Full-length cDNAs, ESTs,
or fragments thereof corresponding to one of the nucleotide
sequences of the present invention, or selected at random from a
cDNA library relevant to the present invention, are arranged on an
appropriate substrate, e.g., a glass slide. The cDNA is fixed to
the slide using, e.g., UV cross-linking followed by thermal and
chemical treatments and subsequent drying. (See, e.g., Schena, M.
et al. (1995) Science 270:467-470; and Shalon, D. et al. (1996)
Genome Res. 6:639-645.) Fluorescent probes are prepared and used
for hybridization to the elements on the substrate. The substrate
is analyzed by procedures described above.
[0224] VIII. Complementary Polynucleotides
[0225] Sequences complementary to the HUTRAN-encoding sequences, or
any parts thereof, are used to detect, decrease, or inhibit
expression of naturally occurring HUTRAN. Although use of
oligonucleotides comprising from about 15 to 30 base pairs is
described, essentially the same procedure is used with smaller or
with larger sequence fragments. Appropriate oligonucleotides are
designed using OLIGO 4.06 software and the coding sequence of
HUTRAN. To inhibit transcription, a complementary oligonucleotide
is designed from the most unique 5' sequence and used to prevent
promoter binding to the coding sequence. To inhibit translation, a
complementary oligonucleotide is designed to prevent ribosomal
binding to the HUTRAN-encoding transcript.
[0226] IX. Expression of HUTRAN
[0227] Expression and purification of HUTRAN is achieved using
bacterial or virus-based expression systems. For expression of
HUTRAN in bacteria, cDNA is subcloned into an appropriate vector
containing an antibiotic resistance gene and an inducible promoter
that directs high levels of cDNA transcription. Examples of such
promoters include, but are not limited to, the trp-lac (tac) hybrid
promoter and the T5 or T7 bacteriophage promoter in conjunction
with the lac operator regulatory element. Recombinant vectors are
transformed into suitable bacterial hosts, e.g., BL21(DE3).
Antibiotic resistant bacteria express HUTRAN upon induction with
isopropyl beta-D-thiogalactopyranoside (IPTG). Expression of HUTRAN
in eukaryotic cells is achieved by infecting insect or mammalian
cell lines with recombinant Autographica californica nuclear
polyhedrosis virus (AcMNPV), commonly known as baculovirus. The
nonessential polyhedrin gene of baculovirus is replaced with cDNA
encoding HUTRAN by either homologous recombination or
bacterial-mediated transposition involving transfer plasmid
intermediates. Viral infectivity is maintained and the strong
polyhedrin promoter drives high levels of cDNA transcription.
Recombinant baculovirus is used to infect Spodoptera frugiperda
(Sf9) insect cells in most cases, or human hepatocytes, in some
cases. Infection of the latter requires additional genetic
modifications to baculovirus. (See Engelhard, E. K. et al. (1994)
Proc. Natl. Acad. Sci. USA 91:3224-3227; Sandig, V. et al. (1996)
Hum. Gene Ther. 7:1937-1945.)
[0228] In most expression systems, HUTRAN is synthesized as a
fusion protein with, e.g., glutathione S-transferase (GST) or a
peptide epitope tag, such as FLAG or 6-His, permitting rapid,
single-step, affinity-based purification of recombinant fusion
protein from crude cell lysates. GST, a 26-kilodalton enzyme from
Schistosoma japonicum, enables the purification of fusion proteins
on immobilized glutathione under conditions that maintain protein
activity and antigenicity (Pharmacia, Piscataway, N.J.). Following
purification, the GST moiety can be proteolytically cleaved from
HUTRAN at specifically engineered sites. FLAG, an 8-amino acid
peptide, enables immunoaffinity purification using commercially
available monoclonal and polyclonal anti-FLAG antibodies (Eastman
Kodak, Rochester, N.Y.). 6-His, a stretch of six consecutive
histidine residues, enables purification on metal-chelate resins
(QIAGEN Inc, Chatsworth, Calif.). Methods for protein expression
and purification are discussed in Ausubel, F. M. et al. (1995 and
periodic supplements) Current Protocols in Molecular Biology, John
Wiley & Sons, New York, N.Y., ch 10, 16. Purified HUTRAN
obtained by these methods can be used directly in the following
activity assay.
[0229] X. Demonstration of HUTRAN Activity
[0230] HUTRAN-1
[0231] HUTRAN-1 activity may be demonstrated by the ability to
convert L-phenylalanine and .alpha.-keto-.gamma.-methiolbutyrate to
phenylpyruvate and L-methionine. (Cooper, A. J. L. and Meister, A.
(1985) Meth. Enzymol. 113:344-349.) The amount of phenylpyruvate
formed is measured. The reaction mixture contains 200 mM
ammediol-HCl buffer (pH 9.0), 10 mM L-phenylalanine, 5 mM
.alpha.-keto-.gamma.-methiolbutyrate, and HUTRAN-1 in a final
volume of 0.1 ml. After incubating at 37.degree. C. for 10 minutes,
0.9 ml of 3.33 M NaOH is added. The absorbance at 322 nm, measured
using a spectrophotometer, is proportional to the phenylpyruvate
formed, and thus to the HUTRAN-1 in the starting sample. The
absorbance due to phenylpyruvate is stable for at least 15
minutes.
[0232] HUTRAN-2
[0233] HUTRAN-2 activity may be demonstrated by the ability to
convert L-glutamate and .alpha.-ketoadipate to .alpha.-aminoadipate
and .alpha.-ketoglutarate. (Nakatani, supra.) The amount of
.alpha.-ketoglutarate formed is measured. The standard assay
contains, in a volume of 0.3 ml, 50 .mu.moles potassium phosphate
buffer (pH 7.5), 20 .mu.g pyridoxal phosphate, 0.5 .mu.moles
.alpha.-ketoadipate, and HUTRAN-2. After a 5 minute incubation at
37.degree. C., the reaction is started by addition of 0.2 ml of 0.1
M potassium L-glutamate and allowed to proceed for 10 minutes at
37.degree. C. The reaction (Reaction 1) is terminated by adding 0.1
ml of 1 M HCl. After neutralization of the mixture with 0.1 ml of 1
M KOH, a 0.3 ml aliquot is taken for the determination of the
presence of .alpha.-ketoglutarate. .alpha.-Ketoglutarate is
estimated by the amount of NADH oxidized in the presence of
NH.sub.4.sup.+ and glutamate dehydrogenase. The estimation of
.alpha.-ketoglutarate is performed in a system consisting of 300
.mu.moles potassium phosphate buffer (pH 7.5), 150 .mu.moles
NH.sub.4Cl, 0.3 .mu.mole NADH, and the neutralized reaction mixture
in a total volume of 3.0 ml. The decrease in absorbance at 340 nm
after the addition of glutamate dehydrogenase, measured using a
spectrophotometer, is proportional to the .alpha.-ketoglutarate
formed in Reaction 1, and thus to the HUTRAN-2 in the starting
sample.
[0234] HUTRAN-3
[0235] HUTRAN-3 activity may be demonstrated by the ability to
methylate hnRNP A1 protein in vitro. (Lin, supra). The reaction
contains 490 ng bacterially expressed recombinant human hnRNP A1,
0.93 .mu.M [.sup.3H]S-adenosyl-L-methionine (2.2 .mu.Ci), 2.0 .mu.g
HUTRAN-3, and buffer (25 mM Tris-HCl, 1 mM EDTA, and 1 mM EGTA at
pH 7.5) in a final volume of 30 .mu.l. The reaction mixtures are
incubated at 30.degree. C. for 30 minutes and then subjected to
SDS-polyacrylamide gel electrophoresis. The gel is stained with
Coomassie Blue, dried and subjected to fluorography. The position
of hnRNP A1 is determined by Coomassie Blue staining. The amount of
[.sup.3H]methylated hnRNP A1, as determined by densitometry or
PHOSPHORIMAGER analysis (Molecular Dynamics, Sunnyvale, Calif.), is
proportional to the amount of HUTRAN-3 in the starting sample.
[0236] XI. Functional Assays
[0237] HUTRAN function is assessed by expressing the sequences
encoding HUTRAN at physiologically elevated levels in mammalian
cell culture systems. cDNA is subcloned into a mammalian expression
vector containing a strong promoter that drives high levels of cDNA
expression. Vectors of choice include PCMV SPORT (Life
Technologies, Gaithersburg, Md.) and PCR 3.1 (Invitrogen, Carlsbad,
Calif., both of which contain the cytomegalovirus promoter. 5-10
.mu.g of recombinant vector are transiently transfected into a
human cell line, preferably of endothelial or hematopoietic origin,
using either liposome formulations or electroporation. 1-2 .mu.g of
an additional plasmid containing sequences encoding a marker
protein are co-transfected. Expression of a marker protein provides
a means to distinguish transfected cells from nontransfected cells
and is a reliable predictor of cDNA expression from the recombinant
vector. Marker proteins of choice include, e.g., Green Fluorescent
Protein (GFP) (Clontech, Palo Alto, Calif.), CD64, or a CD64-GFP
fusion protein. Flow cytometry (FCM), an automated, laser
optics-based technique, is used to identify transfected cells
expressing GFP or CD64-GFP, and to evaluate properties, for
example, their apoptotic state. FCM detects and quantifies the
uptake of fluorescent molecules that diagnose events preceding or
coincident with cell death. These events include changes in nuclear
DNA content as measured by staining of DNA with propidium iodide;
changes in cell size and granularity as measured by forward light
scatter and 90 degree side light scatter; down-regulation of DNA
synthesis as measured by decrease in bromodeoxyuridine uptake;
alterations in expression of cell surface and intracellular
proteins as measured by reactivity with specific antibodies; and
alterations in plasma membrane composition as measured by the
binding of fluorescein-conjugated Annexin V protein to the cell
surface. Methods in flow cytometry are discussed in Ormerod, M. G.
(1994) Flow Cytometry, Oxford, New York, N.Y.
[0238] The influence of HUTRAN on gene expression can be assessed
using highly purified populations of cells transfected with
sequences encoding HUTRAN and either CD64 or CD64-GFP. CD64 and
CD64-GFP are expressed on the surface of transfected cells and bind
to conserved regions of human immunoglobulin G (IgG). Transfected
cells are efficiently separated from nontransfected cells using
magnetic beads coated with either human IgG or antibody against
CD64 (DYNAL, Lake Success, N.Y.). mRNA can be purified from the
cells using methods well known by those of skill in the art.
Expression of mRNA encoding HUTRAN and other genes of interest can
be analyzed by Northern analysis or microarray techniques.
[0239] XII. Production of HUTRAN Specific Antibodies
[0240] HUTRAN substantially purified using polyacrylamide gel
electrophoresis (PAGE)(see, e.g., Harrington, M. G. (1990) Methods
Enzymol. 182:488-495), or other purification techniques, is used to
immunize rabbits and to produce antibodies using standard
protocols.
[0241] Alternatively, the HUTRAN amino acid sequence is analyzed
using LASERGENE software (DNASTAR Inc.) to determine regions of
high immunogenicity, and a corresponding oligopeptide is
synthesized and used to raise antibodies by means known to those of
skill in the art. Methods for selection of appropriate epitopes,
such as those near the C-terminus or in hydrophilic regions are
well described in the art. (See, e.g., Ausubel supra, ch. 11.)
[0242] Typically, oligopeptides 15 residues in length are
synthesized using an Applied Biosystems peptide synthesizer Model
431A using fmoc-chemistry and coupled to KLH (Sigma, St. Louis,
Mo.) by reaction with N-maleimidobenzoyl-N-hydroxysuccinimide ester
(MBS) to increase immunogenicity. (See, e.g., Ausubel supra.)
Rabbits are immunized with the oligopeptide-KLH complex in complete
Freund's adjuvant. Resulting antisera are tested for antipeptide
activity by, for example, binding the peptide to plastic, blocking
with 1% BSA, reacting with rabbit antisera, washing, and reacting
with radio-iodinated goat anti-rabbit IgG.
[0243] XIII. Purification of Naturally Occurring HUTRAN Using
Specific Antibodies
[0244] Naturally occurring or recombinant HUTRAN is substantially
purified by immunoaffinity chromatography using antibodies specific
for HUTRAN. An immunoaffinity column is constructed by covalently
coupling anti-HUTRAN antibody to an activated chromatographic
resin, such as CNBr-activated SEPHAROSE (Pharmacia & Upjohn).
After the coupling, the resin is blocked and washed according to
the manufacturer's instructions.
[0245] Media containing HUTRAN are passed over the immunoaffinity
column, and the column is washed under conditions that allow the
preferential absorbance of HUTRAN (e.g., high ionic strength
buffers in the presence of detergent). The column is eluted under
conditions that disrupt antibody/HUTRAN binding (e.g., a buffer of
pH 2 to pH 3, or a high concentration of a chaotrope, such as urea
or thiocyanate ion), and HUTRAN is collected.
[0246] XIV. Identification of Molecules Which Interact with
HUTRAN
[0247] HUTRAN, or biologically active fragments thereof, are
labeled with .sup.125I Bolton-Hunter reagent. (See, e.g., Bolton et
al. (1973) Biochem. J. 133:529.) Candidate molecules previously
arrayed in the wells of a multi-well plate are incubated with the
labeled HUTRAN, washed, and any wells with labeled HUTRAN complex
are assayed. Data obtained using different concentrations of HUTRAN
are used to calculate values for the number, affinity, and
association of HUTRAN with the candidate molecules.
[0248] Various modifications and variations of the described
methods and systems 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
32 1 454 PRT Homo sapiens misc_feature Incyte ID No 1815528 1 Met
Phe Leu Ala Gln Arg Ser Leu Cys Ser Leu Ser Gly Arg Ala 1 5 10 15
Lys Phe Leu Lys Thr Ile Ser Ser Ser Lys Ile Leu Gly Phe Ser 20 25
30 Thr Ser Ala Lys Met Ser Leu Lys Phe Thr Asn Ala Lys Arg Ile 35
40 45 Glu Gly Leu Asp Ser Asn Val Trp Ile Glu Phe Thr Lys Leu Ala
50 55 60 Ala Asp Pro Ser Val Val Asn Leu Gly Gln Gly Phe Pro Asp
Ile 65 70 75 Ser Pro Pro Thr Tyr Val Lys Glu Glu Leu Ser Lys Ile
Ala Ala 80 85 90 Ile Asp Ser Leu Asn Gln Tyr Thr Arg Gly Phe Gly
His Pro Ser 95 100 105 Leu Val Lys Ala Leu Ser Tyr Leu Tyr Glu Lys
Leu Tyr Gln Lys 110 115 120 Gln Ile Asp Ser Asn Lys Glu Ile Leu Val
Thr Val Gly Ala Tyr 125 130 135 Gly Ser Leu Phe Asn Thr Ile Gln Ala
Leu Ile Asp Glu Gly Asp 140 145 150 Glu Val Ile Leu Ile Val Pro Phe
Tyr Asp Cys Tyr Glu Pro Met 155 160 165 Val Arg Met Ala Gly Ala Thr
Pro Val Phe Ile Pro Leu Arg Ser 170 175 180 Lys Pro Val Tyr Gly Lys
Arg Trp Ser Ser Ser Asp Trp Thr Leu 185 190 195 Asp Pro Gln Glu Leu
Glu Ser Lys Phe Asn Ser Lys Thr Lys Ala 200 205 210 Ile Ile Leu Asn
Thr Pro His Asn Pro Leu Gly Lys Val Tyr Asn 215 220 225 Arg Glu Glu
Leu Gln Val Ile Ala Asp Leu Cys Ile Lys Tyr Asp 230 235 240 Thr Leu
Cys Ile Ser Asp Glu Val Tyr Glu Trp Leu Val Tyr Ser 245 250 255 Gly
Asn Lys His Leu Lys Ile Ala Thr Phe Pro Gly Met Trp Glu 260 265 270
Arg Thr Ile Thr Ile Gly Ser Ala Gly Lys Thr Phe Ser Val Thr 275 280
285 Gly Trp Lys Leu Gly Trp Ser Ile Gly Pro Asn His Leu Ile Lys 290
295 300 His Leu Gln Thr Val Gln Gln Asn Thr Ile Tyr Thr Cys Ala Thr
305 310 315 Pro Leu Gln Glu Ala Leu Ala Gln Ala Phe Trp Ile Asp Ile
Lys 320 325 330 Arg Met Asp Asp Pro Glu Cys Tyr Phe Asn Ser Leu Pro
Lys Glu 335 340 345 Leu Glu Val Lys Arg Asp Arg Met Val Arg Leu Leu
Glu Ser Val 350 355 360 Gly Leu Lys Pro Ile Val Pro Asp Gly Gly Tyr
Phe Ile Ile Ala 365 370 375 Asp Val Ser Leu Leu Asp Pro Asp Leu Ser
Asp Met Lys Asn Asn 380 385 390 Glu Pro Tyr Asp Tyr Lys Phe Val Lys
Trp Met Thr Lys His Lys 395 400 405 Lys Leu Ser Ala Ile Pro Val Ser
Ala Phe Cys Asn Ser Glu Thr 410 415 420 Lys Ser Gln Phe Glu Lys Phe
Val Arg Phe Cys Phe Ile Lys Lys 425 430 435 Asp Ser Thr Leu Asp Ala
Ala Glu Glu Ile Ile Lys Ala Trp Ser 440 445 450 Val Gln Lys Ser 2
425 PRT Homo sapiens misc_feature Incyte ID No 2150892 2 Met Asn
Tyr Ala Arg Phe Ile Thr Ala Ala Ser Ala Arg Arg Asn 1 5 10 15 Pro
Thr Pro Ile Arg Thr Met Thr Asp Ile Leu Ser Arg Gly Pro 20 25 30
Lys Ser Met Ile Ser Leu Ala Gly Gly Leu Pro Asn Pro Asn Met 35 40
45 Phe Pro Phe Lys Thr Ala Val Ile Thr Val Glu Asn Gly Lys Thr 50
55 60 Ile Gln Phe Gly Glu Glu Met Met Lys Arg Ala Leu Gln Tyr Ser
65 70 75 Pro Ser Ala Gly Ile Pro Glu Leu Leu Ser Trp Leu Lys Gln
Leu 80 85 90 Gln Ile Lys Leu His Asn Pro Pro Thr Ile His Tyr Pro
Pro Ser 95 100 105 Gln Gly Gln Met Asp Leu Cys Val Thr Ser Gly Ser
Gln Gln Gly 110 115 120 Leu Cys Lys Val Phe Glu Met Ile Ile Asn Pro
Gly Asp Asn Val 125 130 135 Leu Leu Asp Glu Pro Ala Tyr Ser Gly Thr
Leu Gln Ser Leu His 140 145 150 Pro Leu Gly Cys Asn Ile Ile Asn Val
Ala Ser Asp Glu Ser Gly 155 160 165 Ile Val Pro Asp Ser Leu Arg Asp
Ile Leu Ser Arg Trp Lys Pro 170 175 180 Glu Asp Ala Lys Asn Pro Gln
Lys Asn Thr Pro Lys Phe Leu Tyr 185 190 195 Thr Val Pro Asn Gly Asn
Asn Pro Thr Gly Asn Ser Leu Thr Ser 200 205 210 Glu Arg Lys Lys Glu
Ile Tyr Glu Leu Ala Arg Lys Tyr Asp Phe 215 220 225 Leu Ile Ile Glu
Asp Asp Pro Tyr Tyr Phe Leu Gln Phe Asn Lys 230 235 240 Phe Arg Val
Pro Thr Phe Leu Ser Met Asp Val Asp Gly Arg Val 245 250 255 Ile Arg
Ala Asp Ser Phe Ser Lys Ile Ile Ser Ser Gly Leu Arg 260 265 270 Ile
Gly Phe Leu Thr Gly Pro Lys Pro Leu Ile Glu Arg Val Ile 275 280 285
Leu His Ile Gln Val Ser Thr Leu His Pro Ser Thr Phe Asn Gln 290 295
300 Leu Met Ile Ser Gln Leu Leu His Glu Trp Gly Gly Glu Gly Phe 305
310 315 Met Ala His Val Asp Arg Val Ile Asp Phe Tyr Ser Asn Gln Lys
320 325 330 Asp Ala Ile Leu Ala Ala Ala Asp Lys Trp Leu Thr Gly Leu
Ala 335 340 345 Glu Trp His Val Pro Ala Ala Gly Met Phe Leu Trp Ile
Lys Val 350 355 360 Lys Gly Ile Asn Asp Val Lys Glu Leu Ile Glu Glu
Lys Ala Val 365 370 375 Lys Met Gly Val Leu Met Leu Pro Gly Asn Ala
Phe Tyr Val Asp 380 385 390 Ser Ser Ala Pro Ser Pro Tyr Leu Arg Ala
Ser Phe Ser Ser Ala 395 400 405 Ser Pro Glu Gln Met Asp Val Ala Phe
Gln Val Leu Ala Gln Leu 410 415 420 Ile Lys Glu Ser Leu 425 3 447
PRT Homo sapiens misc_feature Incyte ID No 2525071 3 Met Met Gln
Asp Tyr Val Arg Thr Gly Thr Tyr Gln Arg Ala Ile 1 5 10 15 Leu Gln
Asn His Thr Asp Phe Lys Asp Lys Ile Val Leu Asp Val 20 25 30 Gly
Cys Gly Ser Gly Ile Leu Ser Phe Phe Ala Ala Gln Ala Gly 35 40 45
Ala Arg Lys Ile Tyr Ala Val Glu Ala Ser Thr Met Ala Gln His 50 55
60 Ala Glu Val Leu Val Lys Ser Asn Asn Leu Thr Asp Arg Ile Val 65
70 75 Val Ile Pro Gly Lys Val Glu Glu Val Ser Leu Pro Glu Gln Val
80 85 90 Asp Ile Ile Ile Ser Glu Pro Met Gly Tyr Met Leu Phe Asn
Glu 95 100 105 Arg Met Leu Glu Ser Tyr Leu His Ala Lys Lys Tyr Leu
Lys Pro 110 115 120 Ser Gly Asn Met Phe Pro Thr Ile Gly Asp Val His
Leu Ala Pro 125 130 135 Phe Thr Asp Glu Gln Leu Tyr Met Glu Gln Phe
Thr Lys Ala Asn 140 145 150 Phe Trp Tyr Gln Pro Ser Phe His Gly Val
Asp Leu Ser Ala Leu 155 160 165 Arg Gly Ala Ala Val Asp Glu Tyr Phe
Arg Gln Pro Val Val Asp 170 175 180 Thr Phe Asp Ile Arg Ile Leu Met
Ala Lys Ser Val Lys Tyr Thr 185 190 195 Val Asn Phe Leu Glu Ala Lys
Glu Gly Asp Leu His Arg Ile Glu 200 205 210 Ile Pro Phe Lys Phe His
Met Leu His Ser Gly Leu Val His Gly 215 220 225 Leu Ala Phe Trp Phe
Asp Val Ala Phe Ile Gly Ser Ile Met Thr 230 235 240 Val Trp Leu Ser
Thr Ala Pro Thr Glu Pro Leu Thr His Trp Tyr 245 250 255 Gln Val Arg
Cys Leu Phe Gln Ser Pro Leu Phe Ala Lys Ala Gly 260 265 270 Asp Thr
Leu Ser Gly Thr Cys Leu Leu Ile Ala Asn Lys Arg Gln 275 280 285 Ser
Tyr Asp Ile Ser Ile Val Ala Gln Val Asp Gln Thr Gly Ser 290 295 300
Lys Ser Ser Asn Leu Leu Asp Leu Lys Asn Pro Phe Phe Arg Tyr 305 310
315 Thr Gly Thr Thr Pro Ser Pro Pro Pro Gly Ser His Tyr Thr Ser 320
325 330 Pro Ser Glu Asn Met Trp Asn Thr Gly Ser Thr Tyr Asn Leu Ser
335 340 345 Ser Gly Met Ala Val Ala Gly Met Pro Thr Ala Tyr Asp Leu
Ser 350 355 360 Ser Val Ile Ala Ser Gly Ser Ser Val Gly His Asn Asn
Leu Ile 365 370 375 Pro Leu Ala Asn Thr Gly Ile Val Asn His Thr His
Ser Arg Met 380 385 390 Gly Ser Ile Met Ser Thr Gly Ile Val Gln Gly
Ser Ser Gly Ala 395 400 405 Gln Gly Ser Gly Gly Gly Ser Thr Ser Ala
His Tyr Ala Val Asn 410 415 420 Ser Gln Phe Thr Met Gly Gly Pro Ala
Ile Ser Met Ala Ser Pro 425 430 435 Met Ser Ile Pro Thr Asn Thr Met
His Tyr Gly Ser 440 445 4 1975 DNA Homo sapiens misc_feature Incyte
ID No 1815528 4 caaaaaaaca cccctcatcc agtctcttcc agcctagaga
tcctggccta cccctccgcc 60 aaagcgcgca ctgagtgcaa accccagagt
caatccctgt cccggctccg ccccccgcgt 120 ccgaatcccg cccagccggg
ccctcaagcc cagtcgggac tcgagcctag ggaggcgagg 180 ttcccgcacc
ggatagcatg tttttggccc agaggagcct ctgctctctt agcggtagag 240
caaaattcct gaagacaatt tcttcttcca aaatcctcgg attctctact tctgctaaaa
300 tgtcactgaa attcacaaat gcaaaacgga ttgaaggact tgatagtaat
gtgtggattg 360 aatttaccaa attggctgca gacccttctg ttgtgaatct
tggccaaggc tttccagata 420 tatcccctcc tacatatgta aaagaagaat
tatcaaagat tgcagcaatc gatagcctga 480 atcagtatac acgaggcttt
ggccatccat cacttgtgaa agctctgtcc tatctgtatg 540 aaaagcttta
tcaaaagcaa attgattcaa ataaagaaat ccttgtgaca gtaggagcat 600
atggatctct ttttaacacc attcaagcat taattgatga gggagatgaa gtcatactaa
660 tagtgccttt ctatgactgc tatgagccca tggtgagaat ggctggagca
acacctgttt 720 ttattcccct gagatctaaa cctgtttatg gaaaaagatg
gtctagttct gactggacat 780 tagatcctca agaactggaa agtaaattta
attccaaaac caaagctatt atactaaata 840 ctccacataa cccacttggc
aaggtgtata acagagagga actgcaagta attgctgacc 900 tttgcatcaa
atatgacaca ctctgcatca gtgatgaggt ttatgaatgg cttgtatatt 960
ctggaaataa gcacttaaaa atagctactt ttccaggtat gtgggagaga acaataacaa
1020 taggaagtgc tggaaagact ttcagtgtaa ctggctggaa gcttggctgg
tccattggtc 1080 caaatcattt gataaaacat ttacagacag ttcaacaaaa
cacgatttat acttgtgcaa 1140 ctcctttaca ggaagccttg gctcaagctt
tctggattga catcaagcgc atggatgacc 1200 cagaatgtta ctttaattct
ttgccaaaag agttagaagt aaaaagagat cggatggtac 1260 gtttacttga
aagtgttggc ctaaaaccca tagttcctga tggaggatac ttcatcatcg 1320
ctgatgtgtc tttgctagat ccagacctct ctgatatgaa gaataatgag ccttatgact
1380 ataagtttgt gaaatggatg actaaacata agaaactatc agccatcccc
gtttcagcat 1440 tctgtaactc agagactaaa tcacagtttg agaagtttgt
gcgtttttgc ttcattaaaa 1500 aagacagcac actggatgct gctgaagaaa
tcatcaaggc atggagtgta cagaagtctt 1560 gatttgtgca gaatggatta
atgtttctgt tagatgacct agtatggaat tgttacttag 1620 tgctgccacc
tgctggatgt taaaaggtat ttcagtacaa ctggaattta aatatttcca 1680
ttgtttttcc aaagcagtta acccaactcc taacaacatt ttcgggggat ctgacctttt
1740 ttttccagtt gaaatgtatt aacacacctt ccacaatcat tttataagag
tcagcataac 1800 atagtggata agaactgtga gatgtttaac ctctcagtaa
ctcggttctc tcattataaa 1860 ataggaataa aatcagtacc tgtttcatat
gaaggtcgtt tctgagaatt aaatggacta 1920 atgtatgcaa aaagcctggc
aaacaataaa cactcatctg actttaaaaa aaaaa 1975 5 2125 DNA Homo sapiens
misc_feature Incyte ID No 2150892 5 gcgcgtggga agccagacgc
agcgggggga cacatctcgc ggtggcgttg ccagagtgag 60 gagttagcag
gcaggacttg acgaggctct ttggtttttc tagtcctcaa ccactgaaga 120
agaagcttga tgcttggctg tcagaagaca tgaattacgc acggttcatc acggcagcga
180 gcgcacgcag aaaccctact cccatccgga ccatgactga catattgagc
agaggaccaa 240 aatcgatgat ctccttggct ggtggcttac caaatccaaa
catgtttcct tttaagactg 300 ccgtaatcac tgtagaaaat ggaaagacca
tccaatttgg agaagagatg atgaagagag 360 cacttcagta ttctccgagt
gctggaattc cagagctttt gtcctggcta aaacagttac 420 aaataaaatt
gcataatcct cctaccatcc attacccacc cagtcaagga caaatggatc 480
tatgtgtcac atctggcagc caacaaggtc tttgtaaggt gtttgaaatg atcattaatc
540 ctggagataa tgtcctccta gatgaacctg cttattcagg aactcttcaa
agtctgcacc 600 cactgggctg caacattatt aatgttgcca gtgatgagag
tgggattgtt ccagattccc 660 taagagacat actttccaga tggaaaccag
aagatgcaaa gaatccccag aaaaacaccc 720 ccaaatttct ttatactgtt
ccaaatggca acaaccctac tggaaactca ttaaccagtg 780 aacgcaaaaa
ggaaatctat gagcttgcaa gaaaatatga tttcctcata atagaagatg 840
atccttacta ttttctccag tttaacaagt tcagggtacc aacatttctt tccatggatg
900 ttgatggacg tgtcatcaga gctgactctt tttcaaaaat catttcctct
gggttgagaa 960 taggattttt aactggtcca aaacccttaa tagagagagt
tattttacac atacaagttt 1020 caacattgca ccccagcact tttaaccagc
tcatgatatc acagcttcta cacgaatggg 1080 gaggagaagg tttcatggct
catgtagaca gggttattga tttctatagt aaccagaagg 1140 atgcaatact
ggcagctgca gacaagtggt taactggttt ggcagaatgg catgttcctg 1200
ctgctggaat gtttttatgg attaaagtta aaggcattaa tgatgtaaaa gaactgattg
1260 aagaaaaggc cgttaagatg ggggtattaa tgctccctgg aaatgctttc
tacgtcgata 1320 gctcagctcc tagcccttac ttgagagcat ccttctcttc
agcttctcca gaacagatgg 1380 atgtggcctt ccaggtatta gcacaactta
taaaagaatc tttatgaaga aattaaacta 1440 ggttgggcat ggtggctcac
acctataatc ccagcacttt gggaggcaga ggagggagga 1500 tcacttggac
ccaggaattc aaggctgcag taagctacga tcacaccact gcactctggc 1560
ctgcatgcac tctggcctgc atggcagaac aagaccctgt ctctaaaaaa agagaaagaa
1620 atcaaactaa tcatgctgct catggatttt tccaataaat ttcttgtttt
ggcaggaaga 1680 aatgaacact ggtattagac ttaaagatta aatttcctca
aacatgtcct atctgtagta 1740 gttcaactag acacctttta aagtgcctct
aaattcatca gatggccaaa ctgtatttat 1800 aatccactta ggcattttga
aaaactttca acctgtaaaa agttactttt atcttggatt 1860 tattatgaag
aactttgtag ttgctttgta atttcccata aattgtcttt gaaactaaca 1920
ttttacactg aattattttg agattttaaa gaagtaatta agtgcaaaat ggtatataat
1980 gtgtactttt tctactttta ggaaaattta atgagagctt attgcaaaaa
ttgttataat 2040 ttggtcatta taagtgactt ttagtaaaag taccataaac
cttatgttat gccacagaaa 2100 ttcctttaaa ataaaattct taaat 2125 6 2224
DNA Homo sapiens misc_feature Incyte ID No 2525071 6 cagagtgcag
ccgtgtgggc aagcagtcct tcatcatcac cctgggctgc aacagcgtcc 60
tcatccagtt cgccacaccc aacgattcgg ctcgagcttc tacaacatcc tgaaaacctg
120 ccggggccac accctggagc ggtctgtgtt cagcgagcgg acggaggagt
cttctgccgt 180 gcagtacttc cagttttatg gctacctgtc ccagcagcag
aacatgatgc aggactacgt 240 gcggacaggc acctaccagc gcgccatcct
gcaaaaccac accgacttca aggacaagat 300 cgttcttgat gttggctgtg
gctctgggat cctgtcgttt tttgccgccc aagctggagc 360 acggaaaatc
tacgcggtgg aggccagcac catggcccag cacgctgagg tcttggtgaa 420
gagtaacaac ctgacggacc gcatcgtggt catcccgggc aaggtggagg aggtgtcact
480 ccccgagcag gtggacatca tcatctcgga gcccatgggc tacatgctct
tcaacgagcg 540 catgctggag agctacctcc acgccaagaa gtacctgaag
cccagcggaa acatgtttcc 600 taccattggt gacgtccacc ttgcaccctt
cacggatgaa cagctctaca tggagcagtt 660 caccaaggcc aacttctggt
accagccatc tttccatgga gtggacctgt cggccctccg 720 aggtgccgcg
gtggatgagt atttccggca gcctgtggtg gacacatttg acatccggat 780
cctgatggcc aagtctgtca agtacacggt gaacttctta gaagccaaag aaggagattt
840 gcacaggata gaaatcccat tcaaattcca catgctgcat tcagggctgg
tccacggcct 900 ggctttctgg tttgacgttg ctttcatcgg ctccataatg
accgtgtggc tgtccacagc 960 cccgacagag cccctgaccc actggtacca
ggtgcggtgc ctgttccagt caccactgtt 1020 cgccaaggca ggggacacgc
tctcagggac atgtctgctt attgccaaca aaagacagag 1080 ctacgacatc
agtattgtgg cccaggtgga ccagaccggc tccaagtcca gtaacctcct 1140
ggatctgaaa aaccccttct ttagatacac gggcacaacg ccctcacccc cacccggctc
1200 ccactacaca tctccctcgg aaaacatgtg gaacacgggc agcacctaca
acctcagcag 1260 cgggatggcc gtggcaggga tgccgaccgc ctatgacttg
agcagtgtta ttgccagtgg 1320 ctccagcgtg ggccacaaca acctgattcc
tttagccaac acggggattg tcaatcacac 1380 ccactcccgg atgggctcca
taatgagcac ggggattgtc caagggtcct ccggcgccca 1440 gggcagtggt
ggtggcagca cgagtgccca ctatgcagtc aacagccagt tcaccatggg 1500
cggccccgcc atctccatgg cgtcgcccat gtccatcccg accaacacca tgcactacgg
1560 gagctagggg cccgccccgc ggactgacag caccaggaaa ccaaatgatg
tccctgcccg 1620 ccgcccccgc cgggcggctt tcccccttgt actggagaag
ctcgaacacc cggtcacagc 1680 tctctttgct atgggaactg ggacactttt
ttacacgatg ttgccgccgt ccccacccta 1740 acccccacct cccggccctg
agcgtgtgtc gctgccatat tttacacaaa atcatgttgt 1800 gggagccctc
gtcccccctc ctgcccgctc taccctgacc tgggcttgtc atctgctgga 1860
acaggcgcca tggggcctgc cagccctgcc tgccaggtcc cttagcacct gtccccctgc
1920 ctgtctccag tgggaaggta
gcctggccag gcggggcctc cccttcgacg accaggcctc 1980 ggtcacaacg
gacgtgacat gctgcttttt ttaattttat ttttttatga aaagaaccag 2040
tgtcaatccg cagaccctct gtgaagccag gccggccggg ccgagccagc agcccctctc
2100 cctagactca gaggcgccgc ggggaggggt ggccccgccg aggcttcagg
ggccccctcc 2160 ccaccaaagg gttcacctca cacttgaatg tacaacccac
cccactgtcg ggaaggcctc 2220 cgtc 2224 7 244 DNA Homo sapiens
misc_feature Incyte ID No 1815528H1 7 attatactaa atactccaca
taacccactt ggcaaggtgt ataacagaga ggaactgcaa 60 gtaattgctg
acctttgcat caaatatgac acactctgca tcagtgatga ggtttatgaa 120
tggcttgtat attctggaaa taagcactta aaaatagcta cttttccagg tatgtgggag
180 agaacaataa caataggaag tgctggaaag actttcagtg taactggctg
gaagcttggc 240 tggt 244 8 528 DNA Homo sapiens misc_feature Incyte
ID No 2880980F6 8 attaaagtta cattatctaa aaaaaaaact agaaataact
atactggcta aattataaca 60 cacttatttt cattgaattt atgtatcttt
gttatgtttt tagattatac taaatgtgat 120 taattggaaa acaatattta
cccttttttc ctcctctgtt tagctacttt tccaggtatg 180 tgggagagaa
caataacaat aggaagtgct ggaaagactt tcagtgtaac tggctggaag 240
cttggctggt ccattggtcc aaatcatttg ataaaacatt tacagacagt tcaacaaaac
300 acgatttata cttgtgcaac tcctttacag gaagccttgg ctcaagcttt
ctggattgac 360 atcaagcgca tggatgaccc agaatgttac tttaattctt
tgccaaaagn gttagaagta 420 aaaagagatc ggatggtacg tttacttgaa
aagtgttggg cctaaaaacc catagttccn 480 gganggaggg atacttcatc
atcggctgga tgnggncttt ggccagat 528 9 622 DNA Homo sapiens
misc_feature Incyte ID No 1815528X12C1 9 caatcgatag cctgnaatca
gtatacacga ggctttggcc atccatcact tgtgaaagct 60 ctgtcctatc
tgtatgaaaa gctttatcaa aagcaaattg attcaaataa agaaatcctt 120
gtgacagtag gagcatatgg atctcttttt aacaccattc aagcattaat tgatgaggga
180 gatgaagtca tactaatagt gcctttctat gactgctatg agcccatggt
gagaatggct 240 ggagcaacac ctgtttttat tcccctgaga tctaaacctg
tttatggaaa aagatggtct 300 agttctgact ggacattaga tcctcaagaa
ctggaaagta aatttaattc caaaaccaaa 360 gctattatac taaatactcc
acataaccca cttggcaagg tgtataacag agaggaactg 420 caagtaattg
ctgacctttg catcaaatat gacacactct gattcagtga tgaggtttat 480
gaatggcttg tatattcgga aataagcact aaaaatagct actttccggt atgtgggaga
540 gaacaataac aataggaagt gctggaaaga cttcgtgtaa ctggctggaa
gctgggctgg 600 tccttngtcc aatcattgat aa 622 10 602 DNA Homo sapiens
misc_feature Incyte ID No 1815528X17C1 10 agcattaatt gatgagggag
atgaagtcat actaatagtg cctttctatg actgctatga 60 gcccatggtg
agaatggctg gagcaacacc tgtttttatt cccctgagat ctaaacctgt 120
ttatggaaaa agatggtcta gttctgactg gacattagat cctcaagaac tggaaagtaa
180 atttaattcc aaaaccaaag ctattatact aaatactcca cataacccac
ttggcaaggt 240 gtataacaga gaggaactgc aagtaattgc tgacctttgc
atcaaatatg acacactctg 300 catcagtgat gaggtttatg aatggcttgt
atattctgga aataagcact taaaaatagc 360 tacttttcca ggtatgtggg
agagaacaat aacaatagga agtgctggaa agactttcag 420 tgtaactggg
ctggaagctt ggctggtcca ttggttccaa attctttgat aaaactttac 480
agacgttcaa caaaacacga tttatactgt ggcaacttcc tttacaggaa gcctggctca
540 agcttctgga ttgactcaag cgctggatga cccgaatgtt acttaattct
tgccaaagag 600 ta 602 11 789 DNA Homo sapiens misc_feature Incyte
ID No 1819092T6 11 cnaattttta atttaatatt tggtaagtgt gtggttatat
aattattttc atcataagaa 60 ttatgganaa aatttacaaa tnacaaaaat
ttgtgataat ttaccttttt ctcatactaa 120 atccttcaag gcctcactgt
ctttttcctg gggtcctgga gccttctgac tggcttctct 180 ctgtgcattg
caggacttct cctcaggtca acttatcttc ttntaacatc cancaggtng 240
cagcnctaan taacnantcc atactaggtc atctaacaga aacatnaatc cattctgcan
300 nantcaagac ttctgtacac tccangcctn gatgatttct tcagcancat
ccagtgtgct 360 gnctttttta atgaagcana aacgcncaaa cttctcaaac
tgtgatttag tctctgagtt 420 acagaatgct gaaacgggga tggctgatag
tttcttatgn ttagtcatcc anttnacnaa 480 cttatngtca taaggctcat
tantctcata tcagagatgg ctggatctag caaagtcaca 540 tcagcgatga
tgaagtatcc tccnacagga ctatggggtt taggccacac tntcaagtan 600
acgtaccntc cgngtctnct tactccaact ctttngnaag attaaagtaa cattctgggn
660 caacatgcgc cgatgcatcn agaagctnan ccagggnctg ntnnagatct
aaaaccgaaa 720 gnattggncg tatttngtac ttnngtagaa tggagccggt
aatttggaag agcncccgaa 780 acggatctg 789 12 646 DNA Homo sapiens
misc_feature Incyte ID No 269916F1 12 aaagtcagat gagtgtttat
tgtttgccag gntttttgca tacattagtc catttaattc 60 tcagaaacga
ccttcatatg aaacaggtac tgattttatt cctattttat aatgagagaa 120
ccgagttact gagaggttaa acatctcaca tttcttatcc actatgttat gctgactctt
180 ataaaatgat tgtggaaggt gtgttaatac atttcaactg gaaaaaaaag
gtcagatccc 240 ccgaaaatgt tgttaggagt tgggttaact gctttggaaa
aacaatggaa atatttaaat 300 tccagttgta ctgaaatacc ttttaacatc
cagcaggtgg cagcactaag taacaattcc 360 atactaggtc atctaacaga
aacattaatc cattctgcac aaatcaagac ttctgtacac 420 tccatgcctt
gatgatttct tcagcagcat ccagtgtgct gtctttttta atgaagcaaa 480
aacgcacaaa cttctcaact gtgatttagt ctctgagtta cagantgctg aaacggggtg
540 gctgntagtt tcttatgttt aggccatcca tttcacaaac ttatagncat
aaggctcatt 600 attcttcata tcagngnggt ctgggnctgg caanggcaca tnagcg
646 13 369 DNA Homo sapiens misc_feature Incyte ID No 1717401F6 13
agctagtatg gaattgttac ttagtgctgc cacctgctgg atgttaaaag gnatttcagt
60 acaactggaa tttaaatatt tccattgttt ttccaaagca gttaacccaa
ctcctaacaa 120 cattttcggg ggatctgacc ttttttttcc agttgaaatg
tattaacaca ccttccacaa 180 tcattttata agagtcagca taacatagtg
gataagaact gtgagatgtt taacctctca 240 gtaactcggt tctctcatta
taaaatagga ataaaatcag tacctgtttc atatgaaggt 300 cgtttctgag
aattaaatgg actaatgtat gcaaaaagcc tggcaaacaa taaacactca 360
tctgacttt 369 14 243 DNA Homo sapiens misc_feature Incyte ID No
2150892H1 14 gaccatccaa tttggagaag agatgatgaa gagagcactt cagtattctc
cgagtgctgg 60 aattccagag cttttgtcct ggctaaaaca gttacaaata
aaattgcata atcctcctac 120 catccattac ccacccagtc aaggacaaat
ggatctatgt gtcacatctg gcagccaaca 180 aggtctttgt aaggtgtttg
anatgatcat taatcctgga gataatgtcc tcctagatga 240 acc 243 15 569 DNA
Homo sapiens misc_feature Incyte ID No SAGA00872F1 15 aggcgcgtgg
gaagccagac gcagcggggg gacacatctc gcggtggcgt tgcagagtga 60
ggngttagca ggcaggactt gacgaggctc tttggttttt ctagtcctca accactgaag
120 aagaagcttg atgcttggct gtcagaagac atgaattacg cacggttcat
cacggcagcg 180 agcgcancag aaaccctact cccatccgga ccatgactga
catattgagc agaggaccaa 240 aatcgatgat ctccttggct ggtggcttac
caaatccaaa catgtttcct tttaagactg 300 ccgtaatcac tgtagaaaat
ggaaagacca tccaatttgg agaagagatg atgaagagag 360 cacttcagta
ttctccgagt gctggaattc cagagctttt gtcctggcta aaacagttac 420
aaataaaatt gcataatcct cctaccatcc attaccaccc agtcaaggac aaatggatct
480 atgtgtcaca tctggcagcc aacaaggtct ttgtaaggtg tttgaaatga
tcattaatcc 540 tggagataat gtcctcctag atgaacctg 569 16 526 DNA Homo
sapiens misc_feature Incyte ID No SAGA01877F1 16 ggtcgactct
agaggatccc cccatgaaac cttctcctcc ccattcgtgt agaagctgtg 60
atatcatgag ctggtcaaaa gtgctggggt gcaatgttga aacttgtatg tgtaaaataa
120 ctctctctat taagggtttt ggaccagtta aaaatcctat tctcaaccca
gagggaatga 180 tttttgaaaa agagtcagct ctgatgacac gtccatcaac
atccatggaa agaaatgttg 240 gtaccctgaa cttgttaaac tggagaaaat
agtaaggatc atcttctatt atgaggaaat 300 catattttct tgcaagctca
tagatttcct ttttgcgttc actggttaat gagtttccag 360 tagggttgtt
gccatttgga acagtataaa gaaatttggg ggtgtttttc tggggattct 420
ttgcatcttc tggtttccat ctggaaagta tgtctcttag ggaatctgga acaatcccac
480 tctcatcact ggcaacatta ataatgttgc aggggtaccg agctcg 526 17 467
DNA Homo sapiens misc_feature Incyte ID No SAGA01269R1 17
ttattaatgt tgccagtnat gagagtggga ttgttccaga ttccctaaga gacatacttt
60 ccagatggaa accagaagat gcaaagaatc cccagaaaaa cacccccaaa
tttctttata 120 ctgttccaaa tggcaacaac cctactggaa actcattaac
cagtgaacgc aaaaaggaaa 180 tctatgagct tgcaagaaaa tatgatttcc
tcataataga agatgatcct tactattttc 240 tccagtttaa caagttcagg
gtaccaacat ttctttccat ggatgttgat ggacgtgtca 300 tcagagctga
ctctttttca aaaatcattt cctctgggtt gagaatagga tttttaactg 360
gtccaaaacc cttaatagag agagttattt tacacataca agtttcaaca ttgcacccca
420 gcacttttaa ccagctcatg atatcacagg gggatcctct agagtcg 467 18 338
DNA Homo sapiens misc_feature Incyte ID No SAGA02228F1 18
gcaggtcgac tctagaggat ccccctgcag acaagtggtt aactggtttg gcagaatggc
60 atgttcctgc tgctggaatg tttttatgga ttaaagttaa aggcattaat
gatgtaaaag 120 aactgattga agaaaaggcc gttaagatgg gggtattaat
gctccctgga aatgctttct 180 acgtcgatag ctcagctcct agcccttact
tgagagcatc cttctcttca gcttctccag 240 aacagatgga tgtggccttc
caggtattag cacaacttat aaaagaatct ttatgaagaa 300 attaaactag
gttgggcatg gtgggggtac cgagctcg 338 19 605 DNA Homo sapiens
misc_feature Incyte ID No SAGA01614F1 19 tgcaggtcga ctctagagga
tccccccgtt aagatggggg tattaatgct ccctggaaat 60 gctttctacg
tcgatagctc agctcctagc ccttacttga gagcatcctt ctcttcagct 120
tctccagaac agatggatgt ggccttccag gtattagcac aacttataaa agaatcttta
180 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 240 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 300 nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 360 nnnnnnnnnn nnnnnnnnnn
nnnnnntcat gctgctcatg gatttttcca ataaatttct 420 tgttttggca
ggaagaaatg aacactggta ttagacttaa agattaaatt tcctcaaaca 480
tgtcctattc tgtagnagtt caactagaca ccttttaaag tgcctctaaa ttcatcagat
540 ggccaaactg tatttataat ccacttaggc attttgaaaa acttcaacct
gtaaaaagnt 600 acttt 605 20 495 DNA Homo sapiens misc_feature
Incyte ID No 301251T6 20 gttaagaatt ttattttaaa ggaatttctg
tggcataaca taaggtttat ggtactttta 60 ctaaaagtca cttataatga
ccaaattata acaatttttg caataagctc tcattaaatt 120 ttcctaaaag
tagaaaaagt acacattata taccattttg cacttaatta cttctttaaa 180
atctcaaaat aattcagtgt aaaatgttag tttcaaagac aatttatggg aaattacaaa
240 gcaactacaa agttcttcat aataantcca agataaaagt aactttttac
aggttgaaag 300 tttttcaaaa tgcctaagtg gattataaat acagtttggc
catctgatga atttagaggc 360 actttaaaag gtgtctagtt gaactactac
agataggaca tgtttgagga aatttaatct 420 ttaagtctaa taccagtggt
catttcctcc tgccaaaaca aganatttat tggaaaaatc 480 catgagcagc atgat
495 21 256 DNA Homo sapiens misc_feature Incyte ID No 2525071H1 21
cttctacaac atcctgaaaa cctgccgggg ccacaccctg gagcggtctg tgttcagcga
60 gcggacggag gagtcttctg ccgtgcagta cttccagttt tatggctacc
tgtcccagca 120 gcagaacatg atgcaggact acgtgcggac aggcacctac
cagcgcgcca tcctgcaaaa 180 ccacaccgac ttcaaggaca agatcgttct
tgatgttggc tgtggctctg ggatcctgtc 240 gttttttgcc gcccaa 256 22 258
DNA Homo sapiens misc_feature Incyte ID No 1889292H1 22 cagagtgcag
ccgtgtgggc aagcagtcct tcatcatcac cctgggctgc aacagcgtcc 60
tcatccagtt cgccacaccc aacgatttct gttccttcta caacatcctg aaaacctgcc
120 ggggccacac cctggagcgg tctgtgttca gcgagcggac ggaggagtct
tctgccgtgc 180 agtacttcca gttttatggc tacctgtccc agcagcagaa
catgatgcag gactacgtgc 240 ggacangcac cttaccag 258 23 631 DNA Homo
sapiens misc_feature Incyte ID No 2525071F6 23 cttctacaac
atcctgaaaa cctgccgggg ccacaccctg gagcggtctg tgttcagcga 60
gcggacggag gagtcttctg ccgtgcagta cttccagttt tatggctacc tgtcccagca
120 gcagaacatg atgcaggact acgtgcggac aggcacctac cagcgcggca
tcctgcaaaa 180 ccacaccgac ttcaaggaca agatcgttct tgatgttggc
tgtggctctg ggatcctgtc 240 gttttttgcc gcccaagctg gagcacggaa
aatctacgcg gtggaggcca gcaccatggg 300 cccagcacgc tgaggtcttg
gtgaagagta acaacctgac gggaccgcat cgtggtcatc 360 ccggggcaaa
agtngaagga aggtgtcact tcccccgagc aaggtggaca tcattaatct 420
tgggangccc catggggcnt aanatggntc tttcaaacga agcggcattg ntgggaagaa
480 gctaaccttc cangggccaa agaaaggtaa cttgaaaagn ccccaanccg
ggaaaaaaaa 540 tggtttttcc ctaaaccatt ttgggnggaa ngttcccaac
ntttgggaaa cccctttcaa 600 gngggttgga aaaangtttc ttaaaantng g 631 24
621 DNA Homo sapiens misc_feature Incyte ID No SAEA10009P1 24
atttcngncn tgttgcanaa atgcnnaatt aggacncgga ccccaacgtc ntcatcccgg
60 gcaaggtgga ggaggtgtca ctccccgagc aggtggacat catcatctcg
gagcccatgg 120 gctacatgct cttcaacgag cgcatgctgg anagctacct
ccacgccaag aagtacctga 180 agcccagcgg aaacatgttt cctaccattg
gtgacgtcca ccttgcaccc ttcacggatg 240 aacagctcta catggagcag
ttcaccaagg ccaacttctg gtaccagcca tctttccatg 300 gagtggacct
gtcggccctc cgaggtgccg cnntgnnttn ttntttccgg cagcctgtgg 360
tggacacatt tgacatccgg atcctgatgg ccaagtctgt caagtacacg gtgaacttct
420 tagaagccaa agaaggagat ttgcacagga tanaaatccc attcaaattc
cacatgctgc 480 attcagggct ggtccacggc ctggctttct ggtttgacgt
tgctttcatc ggctccataa 540 tgaccgtgtg gctgtccaca gccccgacag
aacccctgac ccactggtac caggtgcggt 600 gcctgttcca gtcaccactg t 621 25
549 DNA Homo sapiens misc_feature Incyte ID No SAEA03283F1 25
ggctggctgt tgactgcata gtgggcactc gtgctgccac caccactgcc ctgggcgccg
60 gaggaccctt ggacaatccc cgtgctcatt atggagccca tccgggagtg
ggtgtgattg 120 acaatccccg tgttggctaa aggaatcagg ttgttgtggc
ccacgctgga gccactggca 180 ataacactgc tcaagtcata ggcggtcggc
atccctgcca cggccatccc gctgctgagg 240 ttgtaggtgc tgcccgtgtt
ccacatgttt tccgagggag atgtgtagtg ggagccgggt 300 gggggtgagg
gcgttgtgcc cgtgtatcta aagaaggggt ttttcagatc caggaggtta 360
ctggacttgg agccggtctg gtccacctgg gccacaatac tgatgtcgta gctctgtctt
420 ttgttggcaa taagcagaca tgtccctgag agcgtgtccc ctgccttggc
gaacagtggt 480 gactggaaca ggcaccgcac ctggtaccag tgggtcaggg
gctctgtcgg ggctgtggac 540 agccacacg 549 26 647 DNA Homo sapiens
misc_feature Incyte ID No SAEA01931R1 26 ggtggaccag accggctcca
agtccagtaa cctcctggat ctgaaaaacc ccttctttag 60 atacacgggc
acaacgccct cacccccacc cggctcccac tacacatctc cctcggaaaa 120
catgtggaac acgggcagca cctacaacct cagcagcggg atggccgtgg cagggatgcc
180 gaccgcctat gacttgagca gtgttattgc cagtggctcc agcgtgggcc
acaacaacct 240 gattccttta gccaacacgg ggattgtcaa tcacacccac
tcccggatgg gctccataat 300 gagcacgggg attgtccaag ggtcctccgg
cgcccagggc agtggtggtg gcagcacgag 360 tgcccactat gcagtcaaca
gccagttcac catgggcggc cccgccaatc tccatggcgt 420 cgcccatgtc
catcccgacc aacaccatgc actacgggag ctaggggccc gccccgcgga 480
actgacagca ccaggaaacc aaatgatgtc cctgcncgcc gcncccgccg ggcggctttt
540 cccccttgta ctggagaagc tcgaaacaac ccggtcacag ctctctttgc
tatgggaact 600 gggacatttt tttacacgat gttgccgccg tccccaaaac gcgggcg
647 27 655 DNA Homo sapiens misc_feature Incyte ID No 1253024T6 27
ctgccatatt ttacacaaaa tcatgttgtg ggagccctcg ncccccctcc tgcccgctct
60 accctgacct gggcttgtca tctgctggaa caggcgccat ggggcctgcc
agccctgcct 120 gccaggtccc ttagcacctg tcccccngcc ngtctccagt
gggaaggtag cctggccagg 180 cggggcctcc ccttcgacga ccaggcctcg
gtcacaacgg acgtgacatg ctgctttttt 240 taattttatt tttttatgaa
aagaacccag tgtcaatccg cagaccctct gtgaagccag 300 gccggccggg
ccgancaaga ggnccttttc cctagactca gagccncccc ggggaagggg 360
tttccccgcc gaaggttcag ggnngccccc ttcccnacca aaangggttt aacctcaaaa
420 ttnnaaangn aanatcttac cccccattnn tggggaaagg gctnccgntc
cttnngcccc 480 ngnntttttt ggnnnnntnn tttttttccn naanccccng
gaagntcccn nnntttttnt 540 ttnnnnantt aannntttan annnnggnng
gnnaaaggnn tttnggcccc cntggggnaa 600 gnnnnttgng nggcnaattt
ngggggnnaa aaangnnncc nnnaanggnt ttttt 655 28 529 DNA Homo sapiens
misc_feature Incyte ID No 1664573F6 28 tgcctgtctc cagtgggaag
gtagcctggc caggcggggc ctccccttcg acgaccaggc 60 ctcggtcaca
acggacgtga catgctgctt tttttaattt tattttttta tgaaaagaac 120
cagtgtcaat ccgcagaccc tctgtgaagc caggccggcc gggccgagcc agcngcccct
180 ctccctagac tcagaggcgc cgcggggagg ggtnnccccg ccgaggcttc
agggnnnccc 240 tccccaccaa agggttcacc tcacacttga atgtacancc
cancccactg tcgggaaggc 300 tccgtcctcn ncccctgcct cttgctgctg
tcctgtcccc gancccctgc agtcnnctnc 360 ntttnncant naagantaga
gnagtggtgn ngcttgggcc ggaggaaggc atgcggccan 420 tggganaana
gacactcaag attgtaggag ggtctttcct tgagtaagta gctgagagtc 480
cctcatctgn tagtcagtct atatggagga ttcatcctcc tgcggaaga 529 29 220
DNA Homo sapiens misc_feature Incyte ID No 1474156T1 29 gnnccttaat
tttatttttt tatgaaaaga accagtgtca atccgcagac cctctgtgaa 60
gccaggccgg ccgggccgag ccagcagccc ctctccctag actcagaggc gccgcgggga
120 ggggtggccc cgccgaggct tcaggggccc cctccccacc aaagggttca
cctcacactt 180 gaatgtacaa cccaccccac tgtcgggaag gcctccgtcc 220 30
422 PRT Homo sapiens misc_feature GenBank ID No g758591 30 Met Ala
Lys Gln Leu Gln Ala Arg Arg Leu Asp Gly Ile Asp Tyr 1 5 10 15 Asn
Pro Trp Val Glu Phe Val Lys Leu Ala Ser Glu His Asp Val 20 25 30
Val Asn Leu Gly Gln Gly Phe Pro Asp Phe Pro Pro Pro Asp Phe 35 40
45 Ala Val Glu Ala Phe Gln His Ala Val Ser Gly Asp Phe Met Leu 50
55 60 Asn Gln Tyr Thr Lys Thr Phe Gly Tyr Pro Pro Leu Thr Lys Ile
65 70 75 Leu Ala Ser Phe Phe Gly Glu Leu Leu Gly Gln Glu Ile Asp
Pro 80 85 90 Leu Arg Asn Val Leu Val Thr Val Gly Gly Tyr Gly Ala
Leu Phe 95 100 105 Thr Ala Phe Gln Ala Leu Val Asp Glu Gly Asp Glu
Val Ile Ile 110 115 120 Ile Glu Pro Phe Phe Asp Cys Tyr Glu Pro Met
Thr Met Met Ala 125 130 135 Gly Gly Arg Pro Val Phe Val Ser Leu Lys
Pro Gly Pro Ile Gln 140 145 150 Asn Gly Glu Leu Gly Ser Ser Ser Asn
Trp Gln Leu Asp Pro Met 155 160 165 Glu Leu Ala Gly Lys Phe
Thr Ser Arg Thr Lys Ala Leu Val Leu 170 175 180 Asn Thr Pro Asn Asn
Pro Leu Gly Lys Val Phe Ser Arg Glu Glu 185 190 195 Leu Glu Leu Val
Ala Ser Leu Cys Gln Gln His Asp Val Val Cys 200 205 210 Ile Thr Asp
Glu Val Tyr Gln Trp Met Val Tyr Asp Gly His Gln 215 220 225 His Ile
Ser Ile Ala Ser Leu Pro Gly Met Trp Glu Arg Thr Leu 230 235 240 Thr
Ile Gly Ser Ala Gly Lys Thr Phe Ser Ala Thr Gly Trp Lys 245 250 255
Val Gly Trp Val Leu Gly Pro Asp His Ile Met Lys His Leu Arg 260 265
270 Thr Val His Gln Asn Ser Val Phe His Cys Pro Thr Gln Ser Gln 275
280 285 Ala Ala Val Ala Glu Ser Phe Glu Arg Glu Gln Leu Leu Phe Arg
290 295 300 Gln Pro Ser Ser Tyr Phe Val Gln Phe Pro Gln Ala Met Gln
Arg 305 310 315 Cys Arg Asp His Met Ile Arg Ser Leu Gln Ser Val Gly
Leu Lys 320 325 330 Pro Ile Ile Pro Gln Gly Ser Tyr Phe Leu Ile Thr
Asp Ile Ser 335 340 345 Asp Phe Lys Arg Lys Met Pro Asp Leu Pro Gly
Ala Val Asp Glu 350 355 360 Pro Tyr Asp Arg Arg Phe Val Lys Trp Met
Ile Lys Asn Lys Gly 365 370 375 Leu Val Ala Ile Pro Val Ser Ile Phe
Tyr Ser Val Pro His Gln 380 385 390 Lys His Phe Asp His Tyr Ile Arg
Phe Cys Phe Val Lys Asp Glu 395 400 405 Ala Thr Leu Gln Ala Met Asp
Glu Lys Leu Arg Lys Trp Lys Val 410 415 420 Glu Leu 31 425 PRT Homo
sapiens misc_feature GenBank ID No g1050752 31 Met Asn Tyr Ser Arg
Phe Leu Thr Ala Thr Ser Leu Ala Arg Lys 1 5 10 15 Thr Ser Pro Ile
Arg Ala Thr Val Glu Ile Met Ser Arg Ala Pro 20 25 30 Lys Asp Ile
Ile Ser Leu Ala Pro Gly Ser Pro Asn Pro Lys Val 35 40 45 Phe Pro
Phe Lys Ser Ala Val Phe Thr Val Glu Asn Gly Ser Thr 50 55 60 Ile
Arg Phe Glu Gly Glu Met Phe Gln Arg Ala Leu Gln Tyr Ser 65 70 75
Ser Ser Tyr Gly Ile Pro Glu Leu Leu Ser Trp Leu Lys Gln Leu 80 85
90 Gln Ile Lys Leu His Asn Pro Pro Thr Val Asn Tyr Ser Pro Asn 95
100 105 Glu Gly Gln Met Asp Leu Cys Ile Thr Ser Gly Cys Gln Asp Gly
110 115 120 Leu Cys Lys Val Phe Glu Met Leu Ile Asn Pro Gly Asp Thr
Val 125 130 135 Leu Val Asn Glu Pro Leu Tyr Ser Gly Ala Leu Phe Ala
Met Lys 140 145 150 Pro Leu Gly Cys Asn Phe Ile Ser Val Pro Ser Asp
Asp Cys Gly 155 160 165 Ile Ile Pro Glu Gly Leu Lys Lys Val Leu Ser
Gln Trp Lys Pro 170 175 180 Glu Asp Ser Lys Asp Pro Thr Lys Arg Thr
Pro Lys Phe Leu Tyr 185 190 195 Thr Ile Pro Asn Gly Asn Asn Pro Thr
Gly Asn Ser Leu Thr Gly 200 205 210 Asp Arg Lys Lys Glu Ile Tyr Glu
Leu Ala Arg Lys Tyr Asp Phe 215 220 225 Leu Ile Ile Glu Asp Asp Pro
Tyr Tyr Phe Leu Gln Phe Thr Lys 230 235 240 Pro Trp Glu Pro Thr Phe
Leu Ser Met Asp Val Asp Gly Arg Val 245 250 255 Ile Arg Ala Asp Ser
Leu Ser Lys Val Ile Ser Ser Gly Leu Arg 260 265 270 Val Gly Phe Ile
Thr Gly Pro Lys Ser Leu Ile Gln Arg Ile Val 275 280 285 Leu His Thr
Gln Ile Ser Ser Leu His Pro Cys Thr Leu Ser Gln 290 295 300 Leu Met
Ile Ser Glu Leu Leu Tyr Gln Trp Gly Glu Glu Gly Phe 305 310 315 Leu
Ala His Val Asp Arg Ala Ile Asp Phe Tyr Lys Asn Gln Arg 320 325 330
Asp Phe Ile Leu Ala Ala Ala Asp Lys Trp Leu Arg Gly Leu Ala 335 340
345 Glu Trp His Val Pro Lys Ala Gly Met Phe Leu Trp Ile Lys Val 350
355 360 Asn Gly Ile Ser Asp Ala Lys Lys Leu Ile Glu Glu Lys Ala Ile
365 370 375 Glu Arg Glu Ile Leu Leu Val Pro Gly Asn Ser Phe Phe Val
Asp 380 385 390 Asn Ser Ala Pro Ser Ser Phe Phe Arg Ala Ser Phe Ser
Gln Val 395 400 405 Thr Pro Ala Gln Met Asp Leu Val Phe Gln Arg Leu
Ala Gln Leu 410 415 420 Ile Lys Asp Val Ser 425 32 343 PRT Homo
sapiens misc_feature GenBank ID No g1808648 32 Met Glu Val Ser Cys
Gly Gln Ala Glu Ser Ser Glu Lys Pro Asn 1 5 10 15 Ala Glu Asp Met
Thr Ser Lys Asp Tyr Tyr Phe Asp Ser Tyr Ala 20 25 30 His Phe Gly
Ile His Glu Glu Met Leu Lys Asp Glu Val Arg Thr 35 40 45 Leu Thr
Tyr Arg Asn Ser Met Phe His Asn Arg His Leu Phe Lys 50 55 60 Asp
Lys Val Val Leu Asp Val Gly Ser Gly Thr Gly Ile Leu Cys 65 70 75
Met Phe Ala Ala Lys Ala Gly Ala Arg Lys Val Ile Gly Ile Val 80 85
90 Cys Ser Ser Ile Ser Asp Tyr Ala Val Lys Ile Val Lys Ala Asn 95
100 105 Lys Leu Asp His Val Val Thr Ile Ile Lys Gly Lys Val Glu Glu
110 115 120 Val Glu Leu Pro Val Glu Lys Val Asp Ile Ile Ile Ser Glu
Trp 125 130 135 Met Gly Tyr Cys Leu Phe Tyr Glu Ser Met Leu Asn Thr
Val Leu 140 145 150 Tyr Ala Arg Asp Lys Trp Leu Ala Pro Asp Gly Leu
Ile Phe Pro 155 160 165 Asp Arg Ala Thr Leu Tyr Val Thr Ala Ile Glu
Asp Arg Gln Tyr 170 175 180 Lys Asp Tyr Lys Ile His Trp Trp Glu Asn
Val Tyr Gly Phe Asp 185 190 195 Met Ser Cys Ile Lys Asp Val Ala Ile
Lys Glu Pro Leu Val Asp 200 205 210 Val Val Asp Pro Lys Gln Leu Val
Thr Asn Ala Cys Leu Ile Lys 215 220 225 Glu Val Asp Ile Tyr Thr Val
Lys Val Glu Asp Leu Thr Phe Thr 230 235 240 Ser Pro Phe Cys Leu Gln
Val Lys Arg Asn Asp Tyr Val His Ala 245 250 255 Leu Val Ala Tyr Phe
Asn Ile Glu Phe Thr Arg Cys His Lys Arg 260 265 270 Thr Gly Phe Ser
Thr Ser Pro Glu Ser Pro Tyr Thr His Trp Lys 275 280 285 Gln Thr Val
Phe Tyr Met Glu Asp Tyr Leu Thr Val Lys Thr Gly 290 295 300 Glu Glu
Ile Phe Gly Thr Ile Gly Met Arg Pro Asn Ala Lys Asn 305 310 315 Asn
Arg Asp Leu Asp Phe Thr Ile Asp Leu Asp Phe Lys Gly Gln 320 325 330
Leu Cys Glu Leu Ser Cys Ser Thr Asp Tyr Arg Met Arg 335 340
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References